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The regulation of male fertility by the PTPN11 tyrosine phosphatase
Accepted Manuscript Title: The Regulation of Male Fertility by the PTPN11 Tyrosine Phosphatase Author: Pawan Puri William H. Walker PII: DOI: Reference:
S1084-9521(16)30020-9 http://dx.doi.org/doi:10.1016/j.semcdb.2016.01.020 YSCDB 1931
To appear in:
Seminars in Cell & Developmental Biology
Received date: Revised date: Accepted date:
25-11-2015 15-1-2016 18-1-2016
Please cite this article as: Puri Pawan, Walker William H.The Regulation of Male Fertility by the PTPN11 Tyrosine Phosphatase.Seminars in Cell and Developmental Biology http://dx.doi.org/10.1016/j.semcdb.2016.01.020 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
The Regulation of Male Fertility by the PTPN11 Tyrosine Phosphatase
Pawan Puria 1 and William H. Walker a
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Center for Research in Reproductive Physiology, Department of Obstetrics, Gynecology and Reproductive Sciences
Magee Womens Research Institute, University of Pittsburgh, Pittsburgh, PA 15261.
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Present Address: Division of Nephrology, Department of Pediatrics, University of Pittsburgh School of Medicine,
Pittsburgh, PA 15201 USA. Email: [email protected]
Corresponding author: William H. Walker, Ph.D. Department of Obstetrics, Gynecology and Reproductive Sciences Magee Womens Research Institute, University of Pittsburgh 204 Craft Ave, Room B305, Pittsburgh, PA 15261 USA Tel: 412-641-7672 (office) Fax: 412-641-7676 Email: [email protected]
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Graphical Abstract
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Abstract PTPN11 (also known as SHP2) is a ubiquitously expressed non-receptor tyrosine phosphatase that regulates cell survival, proliferation, differentiation, migration and adhesion. Naturally occurring mutations in the PTPN11 gene cause Noonan and LEOPARD syndromes, two genetic disorders that are characterized by a spectrum of defects including male infertility. This review summarizes four cellular and molecular mechanisms by which PTPN11 acts to support male fertility. First, PTPN11 is required for the proliferation and survival of spermatogonial stem cells (SSCs) that are essential to replenish the germ cells that will become sperm. Second, PTPN11 regulation of cellular adhesion functions in Sertoli cells is required to maintain the blood-testis barrier (BTB) that protects meiotic and post-meiotic germ cells. Third, expression of PTPN11 in Sertoli cells is essential to prevent premature differentiation and exhaustion of the SSC population and to maintain the SSC niche. Finally, in Leydig cells, PTPN11 supports mitochondrial fusion and the expression of acyl-CoA synthetase (ACSL4) needed for the production of steroids including testosterone, which is required for fertility.
Abbreviations PTPN11: protein-tyrosine phosphatase non-receptor type 11, SHP2: SH2-containing tyrosine phosphatase 2, SSC: spermatogonial stem cell, ACSL4: acyl-CoA synthetase, GDNF: glial cell-derived neurotrophic factor, bFGF: basic fibroblast growth factor, BTB: blood-testis barrier, FSH: follicle-stimulating hormone, ZBTB16: zinc finger and BTB domain containing 16, PLZF: promyelocytic leukemia zinc finger, CSF-1: colony stimulating factor 1, CXCL12: chemokine (C-X-C motif) ligand 12, STAT3: Signal transducer and activator of transcription 3, SALL4: Spalt-like transcription factor 4, NGN3: Neurogenin-3, SOHLH1/2: Spermatogenesis and oogenesis specific basic helix-loop-helix proteins 1 and 2, ETV5: Ets Variant 5, BCL6B: B-Cell CLL/Lymphoma 6, TER: transepithelial resistance, HGF: hepatic growth factor, FAK: focal adhesion kinase, CSK: C-Src tyrosine kinase, PKA: protein kinase A, STAR: steroidogenic acute regulatory protein, ACOT2: acyl-CoA thioesterase
Keywords Spermatogenesis; stem cell; testis; male fertility; spermatogonia; self-renewal
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1.1 Introduction 1.1.1 Spermatogenesis and supporting cells. Fertility in males is maintained by the process of spermatogenesis that takes place in the seminiferous tubules of the testis and results in the production of more than 100 million sperm daily in men. The seminiferous tubules contain three main cell types; germ cells, epithelial Sertoli cells and peritubular myoid cells. Spermatogonial stem cells (SSC) are the precursors for all germ cells and are considered to be the foundation of this prolific system. SSCs are rare. Approximately 35,000 SSCs are present in a mouse testis comprising about 0.035% of all germ cells [1]. In rodents, stem cell activity is exhibited in a subpopulation of undifferentiated spermatogonia that are present as single cells (As). The As cells can divide to produce separated As cells that retain stem cell activity or they can divide to produce pairs (Apr) and then chains of 4, 8, 16 and 32 cells called Aaligned (Aal) undifferentiated spermatogonia that increasingly lose stem cell activity [2, 3]. The Aal cells proliferate further by mitotic divisions and enter into a differentiation program that results in the formation of preleptotene spermatocytes that undergo meiosis to produce haploid round spermatids that mature and elongate to form spermatozoa. Sertoli cells function to support germ cells at all stages of development and contribute to the SSC niche by secreting glial cell-derived neurotrophic factor (GDNF) and basic fibroblast growth factor (bFGF) that are critical regulators of SSC fate (reviewed in [4]. Specialized Sertoli-Sertoli cell adhesion complexes create the essential bloodtestis-barrier (BTB) that provides a unique environment for meiosis and the development of haploid germ cells. Sertoli cells also provide attachment sites for the developing mitotic, meiotic and postmeiotic germ cells [5]. Peritubular myoid cells lining the exterior of the seminiferous tubules also produce GDNF and contribute to the support of spermatogenesis [6, 7]. Another important cell type present in the interstitium of the testis is the Leydig cell that secretes testosterone, which is essential for the maintenance of spermatogenesis. Macrophages located in the interstitium and intermingled with peritubular myoid cells also have been recently shown to contribute to the SSC niche [8].
1.1.2 PTPN11 structure and function. PTPN11 (protein-tyrosine phosphatase non-receptor type 11) has a classical protein tyrosine phosphatase (PTP) domain and two N-terminal Src homology 2 (SH2) domains [9, 10]. The phosphatase activity of PTPN11 is modulated by a variety of stimuli including hormones, growth factors and cytokines. In the inactive state, access to the PTP domain is blocked by one of the N-terminal SH2 domains [11-13]. After the binding of appropriate ligands to their receptors, auto4
inhibition is relieved due to the recruitment of the SH2 domains to specific phosphotyrosine sites on liganded receptors or receptor-associated adaptor proteins [9, 10]. PTPN11 is an atypical phosphatase because it can have positive or negative regulatory effects on intracellular kinase-mediated signaling pathways [14, 15]. PTPN11 is known to negatively regulate RhoA activity; whereas, PTPN11 can increase or decrease PI3/AKT and JAK/Stat signaling activity [16, 17]. PTPN11 activates the MAPK pathway via well-characterized mechanisms that deactivate upstream inhibitors of Ras. Specifically, activation of PTPN11 results in the dephosphorylation and deactivation of the sprouty protein negative regulator of Ras and the exclusion of p120RasGAP RAS inhibitor from signaling complexes [18, 19]. The PTPN11-mediated modulation of signaling pathways has been shown to regulate cellular proliferation, differentiation, migration and adhesion in numerous cell types. Because of these properties, PTPN11 is a proto-oncogene. Mutations in PTPN1 have been identified in pediatric hematologic malignancies and sold tumors [20-23].
1.1.3 Male infertility in Noonan and LEOPARD syndrome patients having altered PTPN11 activity. A spectrum of PTPN11 gain-of-function or loss-of-function mutations results in Noonan and LEOPARD syndromes in humans, respectively [14, 15]. These syndromes are characterized by short stature, congenital heart and skeleton abnormalities and reproductive defects such as delayed puberty and male infertility [14, 24, 25]. LEOPARD syndrome patients are reported to have abnormal genitalia; however, no information is available describing the causes and mechanisms of infertility in these patients [26]. Men with Noonan syndrome have fertility defects due to cryptorchidism and their testis were reported to have reduced tubular diameter with fewer germ cells and Leydig cells [27]. Noonan syndrome patients have normal testosterone levels but usually have higher follicle-stimulating hormone (FSH) levels [27]. Some Noonan syndrome patients with normal testicular descent have reduced inhibin levels, a possible indicator of compromised Sertoli cell function and maturity [28]. The information derived from Noonan and LEOPARD patients provided the first indication that PTPN11 was required for male fertility.
2.1 Expression and function of PTPN11 in whole testis studies 2.1.1 Expression and localization of PTPN11 in testicular cells. PTPN11 is ubiquitously expressed in somatic cells. Within the testis, immunofluorescence studies revealed that in Sertoli cells, PTPN11 is expressed in the nuclei and cytoplasm of Sertoli cells and appeared to be concentrated in Sertoli 5
cells near the basement membranes of seminiferous tubules in the vicinity of the BTB [29] (Fig. 1A). Expression levels of PTPN11 remain constant in Sertoli cells through all developmental stages from birth to adulthood. In contrast, PTPN11 levels vary dramatically during male germ cell development [29, 30]. PTPN11 is expressed in the most immature germ cells As through Aaligned spermatogonia (Fig. 1B). In these undifferentiated spermatogonia, the phosphatase was detected in the nucleus and cytoplasm as well as at plasma membrane. Detection of PTPN11 at the plasma membrane is consistent with the known interactions of the phosphatase with plasma membrane receptors [31]. Interestingly, the more mature germ cells in the testis are rare examples of cell types that do not express PTPN11. Specifically, differentiated spermatogonia, spermatocytes and spermatids do not show detectable levels of PTPN11 expression. The mechanisms that regulate the differential expression pattern of PTPN11 in undifferentiated vs. differentiated spermatogonia have not been identified.
2.1.2. PTPN11 is required to maintain spermatogenesis. Global deletion of PTPN11 results in embryonic lethality and precludes the examination of PTPN11 function in the mature testis [32]. Therefore, to determine whether PTPN11 is required to maintain spermatogenesis in an adult mouse, a conditional knockout mouse was created by breeding PTPN11 floxed mice to transgenic mice expressing the Cre recombinase fused to a tamoxifen binding ert2 domain and driven by the ubiquitin promoter [33]. The ubiquitous expression pattern the Cre recombinase allowed the deletion of PTPN11 gene in most of the cells of PTPN11floxed ert2 mice that were exposed to tamoxifen. Histological analysis of the adult testis sections showed that 29 days after PTPN11 deletion there was a loss of less mature germ cells, including spermatogonia and spermatocytes [30]. By 43 days after deletion of PTPN11 there was a loss of all germ cells except the most mature elongating spermatids. The remaining elongated spermatids were distributed randomly and in many cases their acrosomes were mis-oriented relative to the Sertoli cells nucleus suggesting that attachment to the Sertoli cell was mis-regulated. Thus far, the direct targets of PTPN11 that are required to maintain proper orientation of elongated spermatids are not known. By 63 days after PTPN11 KO, all germ cells were absent. These studies suggested that knockout of PTPN11 did not directly affect the survival of differentiated spermatogonia and more mature germ cells. However, the lack of PTPN11 appeared to eliminate the undifferentiated spermatogonia that are required to replenish the maturing germ cells [30]. The timing of the progressive loss of germ cells in global PTPN11 KO mice when compared to the length of spermatogenic cycle suggested that there was a defect near the stem cell stage of development. Consistent with this idea, 6
cells expressing the ZBTB16 transcription factor (also known as PLZF), a marker of SSCs and undifferentiated spermatogonia, were not detected in the testis sections after elimination of PTPN11. These results indicated that PTPN11 deletion resulted in the loss of undifferentiated spermatogonia and perhaps SSCs. This initial global PTPN11 KO model showed that PTPN11 is essential for the maintenance of spermatogenesis. However, because PTPN11 was eliminated in all cells, it remained unresolved whether the progressive loss of germ cells was indicative of an intrinsic germ cell requirement for PTPN11 or whether knock out of PTPN11 disrupted the function of cells that contribute to the SSC niche or the hormonal support of spermatogenesis.
3.1. PTPN11 functions in germ cells 3.1.1 PTPN11 is essential to maintain SSCs and undifferentiated spermatogonia. To determine whether PTPN11 expression in germ cells is required to support spermatogenesis, a germ cell specific PTPN11 KO mouse (GCPTPN11KO) was created by mating PTPN11 floxed mice with Vasa Cre mice so that the gene encoding PTPN11 is specifically deleted in gonocytes (prospermatogonia) at embryonic day 15 prior to the formation of SSCs [30]. Histological analysis of testis sections from GCPTPN11KO mice revealed the progressive loss of germ cells in 3 and 4 week-old KO mice and a complete loss of all germ cells by 8 weeks of age. These results provided unambiguous evidence that PTPN11 is required intrinsically in post-natal germ cells to maintain spermatogenesis. PTPN11 does not appear to be essential in gonocytes because the numbers of these cells were not affected in GCPTPN11KO mice [30]. Two populations of gonocytes are known to exist during the first 5 days after birth in mice [34, 35]. One population gives rise to SSCs that are responsible for all the subsequent waves of spermatogenesis throughout life. A second population of gonocytes is thought to be responsible for a one-time bypassing of the SSC and undifferentiated spermatogonia stages of development. These cells differentiate directly into type A1 differentiated spermatogonia resulting in the initial production of germ cells and sperm called the first wave of spermatogenesis. In agreement with the finding that PTPN11 expression is not detected after the undifferentiated spermatogonia stage of germ cell development, the first wave of spermatogenesis occurred normally in the GCPTPN11KO mice. These results indicated that PTPN11 is not essential for maintaining A1 differentiated spermatogonia and later developmental stages of germ cells. In contrast, SSCs and undifferentiated spermatogonia in GCPTPN11KO mice displayed a 50% decrease in proliferation 5 days after birth and a 60% reduction in the number of these cells by 7 days after birth. Increased rates of apoptosis may account for the removal of the remaining SSCs [30]. These findings provided evidence that PTPN11 is 7
essential for the proliferation of SSCs and thus the perpetuation of spermatogenesis. Similar results were observed using cultures of SSCs, as proliferation was decreased after treatment with a PTPN11 inhibitor. Inhibition of PTPN11 in SSC cultures also decreased the activation of ERK that was mediated by the SSC proliferation promoters bFGF and GDNF [30]. Previously studied conditional knockout mouse models revealed that deletion of PTPN11 compromises the proliferation of numerous cell types including neural stem cells, hematopoietic stem cells (HSCs), cardiac progenitors and T cells. In all of these cell types, PTPN11-mediated activation of ERK activation was shown to be a key and common signaling event [36-38].
3.1.2 PTPN11 and SSC renewal. PTPN11 contributes to the renewal of hematopoietic and neural stem cells [36, 39, 40]. It is not yet known whether PTPN11 contributes to maintaining the stem cell qualities (stemness) of SSCs. Transplantation studies are presently the most reliable measure of stem cell activity [41]. However, traditional transplantation studies in which testes lacking germ cells would be the recipients of PTPN11 deficient SSCs are not likely to be informative because PTPN11 is required for the proliferation and expansion of any colonies that would be derived from the transplanted stem cells. In contrast, transplantation assays in which SSCs containing constitutively active PTPN11 are transplanted could determine that PTPN11 expression increases stem cell activity if the number of colonies resulting from transplantation increases (a result of having more cells with stem cell activity). Similarly, an observation that the number of undifferentiated spermatogonia are increased in knock in mouse models harboring gain of function PTPN11 mutations in germ cells would support the hypothesis that PTPN11 contributes to stem cell activity. Naturally occurring mutations in men have provided information regarding the regulation of SSC self-renewal by PTPN11 [42]. In the testes of men in which sporadic PTPN11 gain of function mutations occur during early germ cell development, there was a preferred clonal expansion of SSCs containing the mutation over genotypically normal SSCs [42]. These findings suggest that a mutation that confers increased PTPN11 activity favors SSC self-renewal and expansion by either increasing the proliferation of SSCs or dampening their differentiation.
3.1.3 Defined and potential PTPN11 contributions to signaling pathways in SSCs that regulate self-renewal and differentiation. The self-renewal of SSCs is promoted by the cytokines GDNF, bFGF, colony stimulating factor 1 (CSF-1), and 8
chemokine (C-X-C motif) ligand 12) (CXCL12) [4]. Although the signaling pathways by which these cytokines support self-renewal of SSCs are not well characterized, it is known that GDNF and bFGF activate the AKT and ERK signaling pathways in cultures enriched for SSCs [30, 43-47]. PTPN11 is a known conduit for GDNF- and bFGF-mediated signaling in other cell types [11, 12, 48-52] and PTPN11 activity is required for GDNF or bFGF-mediated induction of ERK phosphorylation in SSC cultures [30] (Fig. 2A). Thus far, it is unknown whether PTPN11 mediates AKT activation by GDNF and/or bFGF in SSCs. Further studies will be required to determine whether PTPN11 contributes to differentially activating the AKT and ERK signaling pathways as a mechanism to modulate SSCs decisions to self-renew versus differentiate. One potential target for PTPN11-mediated regulation of SSC differentiation is a signaling pathway initiated by the regulation of STAT3, a transcription factor known to promote differentiation and inhibit the self-renewal of SSCs [53] (Fig. 2B). PTPN11 is known to directly dephosphorylate and inactivate STAT3 [54-56]. When active, STAT3 stimulates the expression of SALL4 [57] that in turn can sequester ZBTB16 to allow the expression of the transmembrane receptor cKIT that promotes the differentiation of spermatogonia [58]. STAT3 also induces expression of the NGN3 transcription factor that is proposed to interact with E-Box transcription factors (e.g., SOHOH1/2) to drive the expression of genes promoting differentiation [59]. Thus, PTPN11-mediated down-regulation of STAT3 and NGN3 are candidate mechanisms to block the differentiation of SSCs. Other transcription factors that could be downstream targets of PTPN11 in SSCs are ETV5 and BCL6B that are required for SSC self-renewal [60]. These transcription factors are both up-regulated by bFGF and GDNF via the PTPN11-dependent MAP kinase pathway. Notably, expression of ETV5 is severely down regulated after knockout of PTPN11 in the embryonic kidney, plus knockout of ETV5 or PTPN11 produce similar phenotypes such that no germ cells are produced after the first wave [30, 61]
4.1. PTPN11 functions in Sertoli cells 4.1.1 PTPN11 regulates the integrity of the BTB and attachments of SSCs to their niche. The BTB is a specialized junctional complex composed of tight junctions, adherens junctions and gap junctions between adjacent Sertoli cells that physically divides the seminiferous tubules into basal and apical compartments. Disruption of the BTB causes germ cell differentiation and development to be arrested [62]. The BTB undergoes periodic restructuring to allow passage of spermatocytes from the basal to the adluminal compartment of the testis. Kinasemediated signaling cascades are important for maintaining and remodeling the BTB [63]. Changes in the phosphorylation 9
of cell adhesion proteins are known to regulate their localization at sites of adherens and tight junctions [64]. Expression of a constitutively active PTPN11 mutant (SHP2Q79R) disrupted Sertoli-Sertoli cell adhesion in culture as shown by the decrease in transepithelial resistance (TER), a measure of Sertoli cell tight junction and adherens junction integrity [30]. Hepatic growth factor (HGF), a known regulator of BTB function [65] also decreased TER levels and appears to be an upstream regulator of PTPN11 because HGF stimulation of cultured Sertoli cells increased PTPN11-dependent Src and ERK activity. PTPN11 interactions with an HGF receptor (c-Met) adaptor protein (Gab1) previously were shown to be required to mitigate activation of the MAPK pathway [66]. Importantly, altered Src or ERK activity is known to destabilize adherens and tight junctions [63, 67-71]. Studies using cultured rat Sertoli cells suggest that constitutively active PTPN11 acts by decreasing the phosphorylation of paxillin resulting in the dissociation of the negative regulator Csk from Src followed by ERK activation. Furthermore, focal adhesion kinase (FAK), a mediator of cell-cell adhesion, was inactivated and the actin cytoskeleton, required to stabilize cell-cell adhesion, was disrupted in Sertoli cells expressing SHP2Q79R [29]. Finally, the constitutively active PTPN11 mutant caused the mis-localization of the tight junction protein ZO-1, the adapter protein
-catenin and the transmembrane protein N-cadherin away from cell adhesion
sites at the plasma membrane and into the Sertoli cell cytoplasm [29]. PTPN11 also may contribute to anchoring SSCs within their niche adjacent to Sertoli cells. Inhibition of PTPN11 activity in cultures of SSCs resulted in the dispersal of the three dimensional clumps of SSC colonies and their detachment from feeder cells [30]. This result suggests that PTPN11 is required for attachments between the cells within the SSC colonies and between the SSCs and supporting cells. The disruption of cell attachments in the absence of PTPN11 activity in SSCs or Sertoli cells may contribute to the inability to support SSC proliferation and survival in mice lacking PTPN11.
4.1.2 PTPN11 activity in Sertoli cells is required to maintain SSC self-renewal. Additional information regarding PTPN11 functions in Sertoli cells was generated from a transgenic mouse model in which PTPN11 expression was eliminated specifically in Sertoli cells [72]. This model showed that PTPN11 deficiency caused sterility due to an imbalance in the differentiation and self-renewal of SSCs, a disfunctional BTB and the lack of sperm production. In the absence of PTPN11 in Sertoli cells, seminiferous tubules lacked all germ cells and there was Leydig cell hyperplasia but Sertoli cell numbers were not altered. In the PTPN11 knock out mice, the expression of mRNAs encoding junctional proteins associated with the BTB were altered and a representative group of junctional proteins were found to be mis-localized away from the Sertoli cell plasma membrane resulting in a permeable 10
BTB. As found previously [30], expression of a constitutively active PTPN11 mutant in Sertoli cells also impaired tight junction formation between cultured Sertoli cells [72]. The disruption of the BTB is one likely explanation for the loss of meiotic and post-meiotic germ cells in the Sertoli cell-specific PTPN11 knock out mice. Whereas knock out of PTPN11 in germ cells permanently blocked spermatogenesis by inhibiting the proliferation of SSCs, knock out of PTPN11 in Sertoli cells appears to inappropriately promote the differentiation of SSCs [72]. Specifically, the lack of PTPN11 expression in Sertoli cells resulted in rapidly decreasing numbers of SSC-derived undifferentiated spermatogonia beginning two weeks after birth and the premature appearance of cells having markers of differentiated spermatogonia. Also, the expression of genes encoding differentiation-promoting factors was up-regulated in PTPN11 deficient Sertoli cells although it is not yet clear whether the expression of genes promoting SSC renewal are altered by PTPN11 knock out. Together, these findings suggest that the lack of PTPN11 in Sertoli cells halts the replenishment of germ cells by inducing the differentiation of SSCs that results in the depletion of the SSC population. The requirement for PTPN11 to support SSC renewal was confirmed by the finding that cultures enriched for SSCs and undifferentiated spermatogonia could not be supported by Sertoli cells either lacking PTPN11 or expressing a constitutively active PTPN11. These results indicate that “normal” levels of PTPN11 activity in Sertoli cells are required to maintain the balance of renewal and differentiation of SSCs that in turn are essential for the continuation of spermatogenesis and fertility.
5.1 PTPN11 regulates steroidogenesis in Leydig cells. Leydig cells in the testis interstitium initiate testosterone synthesis when activated by luteinizing hormone (LH) secreted from the pituitary. LH stimulation of Leydig cells increases cAMP levels that activates protein kinase A (PKA) and stimulates a rate-limiting step of steroid synthesis pathway, the transport of cholesterol into the mitochondria, by the up-regulation of a transporter, steroidogenic acute regulatory protein (STAR). The expression of STAR is increased by the production of lipoxygenated or epoxygeneted metabolites of aracadonic acid that are regulated by acyl-CoA synethease (ACSL4) and acyl-CoA thioesterase (ACOT2) [73, 74]. A recent study showed that PTPN11 is a critical regulator of the expression levels of ACSL4 in Leydig cells [73]. Treatment of primary Leydig cell cultures with the PTPN11 inhibitor NSC-87877 decreased cAMP-stimulated testosterone production. Furthermore, decreased steroid production after shRNA-mediated PTPN11 knock down is associated with decreased ACSL4 and StAR protein levels; whereas, PTPN11 overexpression increased ACSL4 and STAR protein levels and steroid production. In addition, mitochondrial fusion, a 11
cAMP-stimulated process that is essential for steroid production was inhibited by shRNA-mediated knockdown of PTPN11 [75]. Together, these data show PTPN11 activation is a key step in the activation of cAMP-PKA pathway stimulated steroid production. Further studies are required to determine whether deletion of PTPN11 specifically in Leydig cells impairs testosterone production and/or male fertility.
6.1 Summary and perspectives 6.1.1 Summary PTPN11 is a critical mediator of functions that are required to maintain spermatogenesis and male fertility. In Sertoli cells, PTPN11 is essential to support the SSC niche and germ cell development. Specifically, PTPN11 expression is required to limit the expression of factors that promote the differentiation of SSCs. Correct levels of PTPN11 activity in Sertoli cells also are required to maintain the BTB and the attachment of SSCs and more mature germ cells. In addition, there is evidence that PTPN11 activity is required in Leydig cells to support the production of steroids including testosterone. Finally, PTPN11 expression in SSCs is essential to maintain the stem cell population that replenishes the germ cells that will become sperm. In the future, defining the signaling pathways regulated by PTPN11 in SSCs could provide important information needed to elucidate the mechanisms by which SSCs determine whether they will selfrenew or differentiate.
6.2.1 Potential regulatory mechanisms of PTPN11 expression and targets in SSCs. Although PTPN11 was found to be required in undifferentiated spermatogoia and Sertoli cells to maintain spermatogenesis and fertility, the molecular mechanisms by which PTPN11 expression and activity are regulated require further examination. For example, PTPN11 is expressed in SSCs and undifferentiated spermatogonia, but PTPN11 cannot be detected in differentiated spermatogonia and more mature germ cells. What is the mechanism by which PTPN11 expression is uniquely shut off in the more differentiated spermatogonia? Does the loss of PTPN11 contribute to the differentiation of spermatogonia? Could this shut off mechanism be useful to slow the progression of tumors that are dependent upon PTPN11? The expression of factors that promote the self-renewal of SSCs including GFRA1 and ZBTB16 decrease in tandem with PTPN11 as the spermatogonia differentiate. Does PTPN11 support the expression of proteins that promote self-renewal as well as mediate intracellular signals associated with self-renewal?
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6.3.1 PTPN11 regulation of oxidative phosphorylation and ROS as a putative mechanism for maintaining SSC selfrenewal Self-renewal and proliferation of SSCs is promoted by reactive oxygen species (ROS) that are generated via activation of ERK and AKT after stimulation of the GFRA1, FGFR receptors [76]. Might PTPN11 activation of ERK and AKT contribute to the generation of ROS resulting in increased proliferation and promoting the self-renewal of SSCs? In other cell types, PTPN11 has been localized to the mitochondria [77, 78] raising the possibility that PTPN11 may act directly to alter oxidative phosphorylation and ROS production in SSCs and Sertoli cells. In fact, normal levels of PTPN11 activity in the mitochondria are proposed to keep ROS levels lower in the mitochondria, decrease DNA damage and slow the aging process [79], which are properties that would promote the fidelity and longevity of SSCs. One hypothesis that would unify the information available regarding PTPN11 and ROS production is that in SSCs, PTPN11 in the mitochondria acts to limit ROS production and inhibits aging, but when signaling through the GFRA1 and FGF receptors increases PTPN11 in the cytoplasm proliferation promoting signaling pathways are activated resulting in temporarily elevated ROS production.
6.4.1 Transcriptional regulation by PTPN11 may promote self-renewal PTPN11 has also been localized to the nucleus and acts to regulate transcription by interacting directly with the DNA bound signal transducer and activator of transcription 5 (STAT5) or by dephosphorylating and inactivating STAT1. [80, 81]. Interestingly PTPN11 also has been shown to directly dephosphorylate and inactivate STAT3 [55, 56], which in its active state enhances the expression of factors that promote the differentiation of SSCs [57, 59]. Additional studies will be required to determine whether PTPN11 mediates the inactivation of STAT3 in SSCs and acts to support their selfrenewal.
6.5.1 Future benefits from studies of PTPN11 in the testes The specific mechanisms by which PTPN11 support male fertility remain to be fully revealed. However, because it was found that PTPN11 activity is essential to maintain spermatogenesis, at least one practical application can be envisioned from the advances in understanding the functions of PTPN11. Specifically, inhibitors of PTPN11 activity delivered to the testes could be used to eliminate SSCs and permanently halt sperm production. In fact, an inhibitor of PTPN11 was found to eliminate SSCs and more mature germ cells in mouse testes [30], The development of a sterilant 13
strategy using PTPN11 inhibitors could be used to control animal populations, replace neutering surgeries or used as a replacement for vasectomies. Other possible contributions from PTPN11 studies might be improved strategies to more efficiently culture SSCs for further research or for transplantation into germ cell deficient testes to restore fertility after chemotherapy. Finally, a better understanding of the factors regulated by PTPN11 may facilitate the production of therapies for infertility conditions caused by disruption of SHP2 regulated pathways.
Acknowledgement We thank Dr. Anthony Zeleznik for assistance in the editing of the manuscript.
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References
[1]. R. A. Tegelenbosch, and D. G. de Rooij. A quantitative study of spermatogonial multiplication and stem cell renewal in the C3H/101 F1 hybrid mouse. Mutat Res 1993. 290:193-200. [2]. T. Nakagawa, M. Sharma, Y. Nabeshima, R. E. Braun, and S. Yoshida. Functional hierarchy and reversibility within the murine spermatogenic stem cell compartment. Science 2010. 328:62-7. [3]. K. Hara, T. Nakagawa, H. Enomoto, M. Suzuki, M. Yamamoto, B. D. Simons, et al. Mouse spermatogenic stem cells continually interconvert between equipotent singly isolated and syncytial states. Cell Stem Cell 2014. 14:658-72. [4]. S. R. Chen, and Y. X. Liu. Regulation of spermatogonial stem cell self-renewal and spermatocyte meiosis by Sertoli cell signaling. Reproduction 2015. 149:R159-67. [5]. C. Y. Cheng, and D. D. Mruk. The blood-testis barrier and its implications for male contraception. Pharmacol Rev 2012. 64:16-64. [6]. L. Y. Chen, P. R. Brown, W. B. Willis, and E. M. Eddy. Peritubular myoid cells participate in male mouse spermatogonial stem cell maintenance. Endocrinology 2014. 155:4964-74. [7]. M. Welsh, L. Moffat, L. Jack, A. McNeilly, D. Brownstein, P. T. Saunders, et al. Deletion of androgen receptor in the smooth muscle of the seminal vesicles impairs secretory function and alters its responsiveness to exogenous testosterone and estradiol. Endocrinology 2010. 151:3374-85. [8]. T. DeFalco, S. J. Potter, A. V. Williams, B. Waller, M. J. Kan, and B. Capel. Macrophages Contribute to the Spermatogonial Niche in the Adult Testis. Cell Rep 2015. 12:1107-19. [9]. D. Barford, and B. G. Neel. Revealing mechanisms for SH2 domain mediated regulation of the protein tyrosine phosphatase SHP-2. Structure 1998. 6:249-54. [10]. P. Hof, S. Pluskey, S. Dhe-Paganon, M. J. Eck, and S. E. Shoelson. Crystal structure of the tyrosine phosphatase SHP-2. Cell 1998. 92:441-50. [11]. M. Perrinjaquet, M. Vilar, and C. F. Ibanez. Protein-tyrosine phosphatase SHP2 contributes to GDNF neurotrophic activity through direct binding to phospho-Tyr687 in the RET receptor tyrosine kinase. J Biol Chem 2010. 285:31867-75. [12]. H. Li, C. Tao, Z. Cai, K. Hertzler-Schaefer, T. N. Collins, F. Wang, et al. Frs2alpha and Shp2 signal independently of Gab to mediate FGF signaling in lens development. J Cell Sci 2014. 127:571-82. [13]. J. G. Bode, J. Schweigart, J. Kehrmann, C. Ehlting, F. Schaper, P. C. Heinrich, et al. TNF-alpha induces tyrosine phosphorylation and recruitment of the Src homology protein-tyrosine phosphatase 2 to the gp130 signal-transducing subunit of the IL-6 receptor complex. J Immunol 2003. 171:257-66. [14]. M. Tajan, A. de Rocca Serra, P. Valet, T. Edouard, and A. Yart. SHP2 sails from physiology to pathology. Eur J Med Genet 2015. 58:509-25. [15]. K. S. Grossmann, M. Rosario, C. Birchmeier, and W. Birchmeier. The tyrosine phosphatase Shp2 in development and cancer. Adv Cancer Res 2010. 106:53-89. [16]. S. Q. Zhang, W. G. Tsiaras, T. Araki, G. Wen, L. Minichiello, R. Klein, et al. Receptor-specific regulation of phosphatidylinositol 3'-kinase activation by the protein tyrosine phosphatase Shp2. Mol Cell Biol 2002. 22:4062-72. [17]. Y. Ke, J. Lesperance, E. E. Zhang, E. A. Bard-Chapeau, R. G. Oshima, W. J. Muller, et al. Conditional deletion of Shp2 in the mammary gland leads to impaired lobulo-alveolar outgrowth and attenuated Stat5 activation. J Biol Chem 2006. 281:34374-80. [18]. Y. M. Agazie, and M. J. Hayman. Molecular mechanism for a role of SHP2 in epidermal growth factor receptor signaling. Mol Cell Biol 2003. 23:7875-86. [19]. H. Hanafusa, S. Torii, T. Yasunaga, K. Matsumoto, and E. Nishida. Shp2, an SH2-containing proteintyrosine phosphatase, positively regulates receptor tyrosine kinase signaling by dephosphorylating and inactivating the inhibitor Sprouty. J Biol Chem 2004. 279:22992-5. [20]. R. J. Chan, and G. S. Feng. PTPN11 is the first identified proto-oncogene that encodes a tyrosine phosphatase. Blood 2007. 109:862-7.
15
[21]. M. Tartaglia, C. M. Niemeyer, A. Fragale, X. Song, J. Buechner, A. Jung, et al. Somatic mutations in PTPN11 in juvenile myelomonocytic leukemia, myelodysplastic syndromes and acute myeloid leukemia. Nat Genet 2003. 34:148-50. [22]. M. L. Loh, S. Vattikuti, S. Schubbert, M. G. Reynolds, E. Carlson, K. H. Lieuw, et al. Mutations in PTPN11 implicate the SHP-2 phosphatase in leukemogenesis. Blood 2004. 103:2325-31. [23]. M. Bentires-Alj, J. G. Paez, F. S. David, H. Keilhack, B. Halmos, K. Naoki, et al. Activating mutations of the noonan syndrome-associated SHP2/PTPN11 gene in human solid tumors and adult acute myelogenous leukemia. Cancer Res 2004. 64:8816-20. [24]. A. A. Jorge, A. C. Malaquias, I. J. Arnhold, and B. B. Mendonca. Noonan syndrome and related disorders: a review of clinical features and mutations in genes of the RAS/MAPK pathway. Horm Res 2009. 71:185-93. [25]. I. van der Burgt, E. Berends, E. Lommen, S. van Beersum, B. Hamel, and E. Mariman. Clinical and molecular studies in a large Dutch family with Noonan syndrome. Am J Med Genet 1994. 53:187-91. [26]. E. Nemes, K. Farkas, B. Kocsis-Deak, A. Drubi, A. Sulak, K. Tripolszki, et al. Phenotypical diversity of patients with LEOPARD syndrome carrying the worldwide recurrent p.Tyr279Cys PTPN11 mutation. Arch Dermatol Res 2015. 307:891-5. [27]. I. Sasagawa, T. Nakada, Y. Kubota, T. Sawamura, T. Tateno, and M. Ishigooka. Gonadal function and testicular histology in Noonan's syndrome with bilateral cryptorchidism. Arch Androl 1994. 32:135-40. [28]. K. A. Marcus, C. G. Sweep, I. van der Burgt, and C. Noordam. Impaired Sertoli cell function in males diagnosed with Noonan syndrome. J Pediatr Endocrinol Metab 2008. 21:1079-84. [29]. P. Puri, and W. H. Walker. The tyrosine phosphatase SHP2 regulates Sertoli cell junction complexes. Biol Reprod 2013. 88:59. [30]. P. Puri, B. T. Phillips, H. Suzuki, K. E. Orwig, A. Rajkovic, P. E. Lapinski, et al. The transition from stem cell to progenitor spermatogonia and male fertility requires the SHP2 protein tyrosine phosphatase. Stem Cells 2014. 32:741-53. [31]. Y. Fujioka, T. Matozaki, T. Noguchi, A. Iwamatsu, T. Yamao, N. Takahashi, et al. A novel membrane glycoprotein, SHPS-1, that binds the SH2-domain-containing protein tyrosine phosphatase SHP-2 in response to mitogens and cell adhesion. Mol Cell Biol 1996. 16:6887-99. [32]. T. M. Saxton, M. Henkemeyer, S. Gasca, R. Shen, D. J. Rossi, F. Shalaby, et al. Abnormal mesoderm patterning in mouse embryos mutant for the SH2 tyrosine phosphatase Shp-2. EMBO J 1997. 16:2352-64. [33]. T. J. Bauler, N. Kamiya, P. E. Lapinski, E. Langewisch, Y. Mishina, J. E. Wilkinson, et al. Development of severe skeletal defects in induced SHP-2-deficient adult mice: a model of skeletal malformation in humans with SHP-2 mutations. Dis Model Mech 2011. 4:228-39. [34]. P. M. Kluin, and D. G. de Rooij. A comparison between the morphology and cell kinetics of gonocytes and adult type undifferentiated spermatogonia in the mouse. Int J Androl 1981. 4:475-93. [35]. S. Yoshida, M. Sukeno, T. Nakagawa, K. Ohbo, G. Nagamatsu, T. Suda, et al. The first round of mouse spermatogenesis is a distinctive program that lacks the self-renewing spermatogonia stage. Development 2006. 133:1495-505. [36]. G. Chan, L. S. Cheung, W. Yang, M. Milyavsky, A. D. Sanders, S. Gu, et al. Essential role for Ptpn11 in survival of hematopoietic stem and progenitor cells. Blood 2011. 117:4253-61. [37]. Y. G. Langdon, S. C. Goetz, A. E. Berg, J. T. Swanik, and F. L. Conlon. SHP-2 is required for the maintenance of cardiac progenitors. Development 2007. 134:4119-30. [38]. T. V. Nguyen, Y. Ke, E. E. Zhang, and G. S. Feng. Conditional deletion of Shp2 tyrosine phosphatase in thymocytes suppresses both pre-TCR and TCR signals. J Immunol 2006. 177:5990-6. [39]. H. H. Zhu, K. Ji, N. Alderson, Z. He, S. Li, W. Liu, et al. Kit-Shp2-Kit signaling acts to maintain a functional hematopoietic stem and progenitor cell pool. Blood 2011. 117:5350-61. [40]. Y. Ke, E. E. Zhang, K. Hagihara, D. Wu, Y. Pang, R. Klein, et al. Deletion of Shp2 in the brain leads to defective proliferation and differentiation in neural stem cells and early postnatal lethality. Mol Cell Biol 2007. 27:6706-17. [41]. J. M. Oatley, and R. L. Brinster. Spermatogonial stem cells. Methods Enzymol 2006. 419:259-82. 16
[42]. S. R. Yoon, S. K. Choi, J. Eboreime, B. D. Gelb, P. Calabrese, and N. Arnheim. Age-dependent germline mosaicism of the most common noonan syndrome mutation shows the signature of germline selection. Am J Hum Genet 2013. 92:917-26. [43]. L. Braydich-Stolle, N. Kostereva, M. Dym, and M. C. Hofmann. Role of Src family kinases and N-Myc in spermatogonial stem cell proliferation. Dev Biol 2007. 304:34-45. [44]. J. M. Oatley, M. R. Avarbock, and R. L. Brinster. Glial cell line-derived neurotrophic factor regulation of genes essential for self-renewal of mouse spermatogonial stem cells is dependent on Src family kinase signaling. J Biol Chem 2007. 282:25842-51. [45]. J. Lee, M. Kanatsu-Shinohara, K. Inoue, N. Ogonuki, H. Miki, S. Toyokuni, et al. Akt mediates selfrenewal division of mouse spermatogonial stem cells. Development 2007. 134:1853-9. [46]. Z. He, J. Jiang, M. Kokkinaki, N. Golestaneh, M. C. Hofmann, and M. Dym. Gdnf upregulates c-Fos transcription via the Ras/Erk1/2 pathway to promote mouse spermatogonial stem cell proliferation. Stem Cells 2008. 26:266-78. [47]. K. Ishii, M. Kanatsu-Shinohara, S. Toyokuni, and T. Shinohara. FGF2 mediates mouse spermatogonial stem cell self-renewal via upregulation of Etv5 and Bcl6b through MAP2K1 activation. Development 2012. 139:1734-43. [48]. R. Willecke, J. Heuberger, K. Grossmann, O. Michos, K. Schmidt-Ott, K. Walentin, et al. The tyrosine phosphatase Shp2 acts downstream of GDNF/Ret in branching morphogenesis of the developing mouse kidney. Dev Biol 2011. 360:310-7. [49]. A. D'Alessio, D. Califano, M. Incoronato, G. Santelli, T. Florio, G. Schettini, et al. The tyrosine phosphatase Shp-2 mediates intracellular signaling initiated by Ret mutants. Endocrinology 2003. 144:4298305. [50]. Z. Ahmed, C. C. Lin, K. M. Suen, F. A. Melo, J. A. Levitt, K. Suhling, et al. Grb2 controls phosphorylation of FGFR2 by inhibiting receptor kinase and Shp2 phosphatase activity. J Cell Biol 2013. 200:493-504. [51]. Y. Pan, C. Carbe, A. Powers, E. E. Zhang, J. D. Esko, K. Grobe, et al. Bud specific N-sulfation of heparan sulfate regulates Shp2-dependent FGF signaling during lacrimal gland induction. Development 2008. 135:301-10. [52]. J. Burks, and Y. M. Agazie. Modulation of alpha-catenin Tyr phosphorylation by SHP2 positively effects cell transformation induced by the constitutively active FGFR3. Oncogene 2006. 25:7166-79. [53]. J. M. Oatley, A. V. Kaucher, M. R. Avarbock, and R. L. Brinster. Regulation of mouse spermatogonial stem cell differentiation by STAT3 signaling. Biol Reprod 2010. 83:427-33. [54]. E. E. Zhang, E. Chapeau, K. Hagihara, and G. S. Feng. Neuronal Shp2 tyrosine phosphatase controls energy balance and metabolism. Proc Natl Acad Sci U S A 2004. 101:16064-9. [55]. Y. Yang, B. Jiang, Y. Huo, L. Primo, J. S. Dahl, T. L. Benjamin, et al. Shp2 suppresses PyMT-induced transformation in mouse fibroblasts by inhibiting Stat3 activity. Virology 2011. 409:204-10. [56]. D. J. Kim, M. L. Tremblay, and J. Digiovanni. Protein tyrosine phosphatases, TC-PTP, SHP1, and SHP2, cooperate in rapid dephosphorylation of Stat3 in keratinocytes following UVB irradiation. PLoS One 2010. 5:e10290. [57]. J. D. Bard, P. Gelebart, H. M. Amin, L. C. Young, Y. Ma, and R. Lai. Signal transducer and activator of transcription 3 is a transcriptional factor regulating the gene expression of SALL4. FASEB J 2009. 23:140514. [58]. R. M. Hobbs, S. Fagoonee, A. Papa, K. Webster, F. Altruda, R. Nishinakamura, et al. Functional antagonism between Sall4 and Plzf defines germline progenitors. Cell Stem Cell 2012. 10:284-98. [59]. A. V. Kaucher, M. J. Oatley, and J. M. Oatley. NEUROG3 is a critical downstream effector for STAT3regulated differentiation of mammalian stem and progenitor spermatogonia. Biol Reprod 2012. 86:164, 1-11. [60]. J. A. Schmidt, M. R. Avarbock, J. W. Tobias, and R. L. Brinster. Identification of glial cell line-derived neurotrophic factor-regulated genes important for spermatogonial stem cell self-renewal in the rat. Biol Reprod 2009. 81:56-66.
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[61]. G. Tyagi, K. Carnes, C. Morrow, N. V. Kostereva, G. C. Ekman, D. D. Meling, et al. Loss of Etv5 decreases proliferation and RET levels in neonatal mouse testicular germ cells and causes an abnormal first wave of spermatogenesis. Biol Reprod 2009. 81:258-66. [62]. C. H. Wong, and C. Y. Cheng. The blood-testis barrier: its biology, regulation, and physiological role in spermatogenesis. Curr Top Dev Biol 2005. 71:263-96. [63]. C. H. Wong, and C. Y. Cheng. Mitogen-activated protein kinases, adherens junction dynamics, and spermatogenesis: a review of recent data. Dev Biol 2005. 286:1-15. [64]. J. C. Li, D. Mruk, and C. Y. Cheng. The inter-Sertoli tight junction permeability barrier is regulated by the interplay of protein phosphatases and kinases: an in vitro study. J Androl 2001. 22:847-56. [65]. A. Catizone, G. Ricci, M. Caruso, F. Ferranti, R. Canipari, and M. Galdieri. Hepatocyte growth factor (HGF) regulates blood-testis barrier (BTB) in adult rats. Mol Cell Endocrinol 2012. 348:135-146. [66]. U. Schaeper, N. H. Gehring, K. P. Fuchs, M. Sachs, B. Kempkes, and W. Birchmeier. Coupling of Gab1 to c-Met, Grb2, and Shp2 mediates biological responses. J Cell Biol 2000. 149:1419-32. [67]. R. M. Ray, R. J. Vaidya, and L. R. Johnson. MEK/ERK regulates adherens junctions and migration through Rac1. Cell Motil Cytoskeleton 2007. 64:143-56. [68]. S. A. Woodcock, C. Rooney, M. Liontos, Y. Connolly, V. Zoumpourlis, A. D. Whetton, et al. SRCinduced disassembly of adherens junctions requires localized phosphorylation and degradation of the rac activator tiam1. Mol Cell 2009. 33:639-53. [69]. N. P. Lee, and C. Y. Cheng. Protein kinases and adherens junction dynamics in the seminiferous epithelium of the rat testis. J Cell Physiol 2005. 202:344-60. [70]. Y. Wang, J. Zhang, X. J. Yi, and F. S. Yu. Activation of ERK1/2 MAP kinase pathway induces tight junction disruption in human corneal epithelial cells. Exp Eye Res 2004. 78:125-36. [71]. J. H. Lipschutz, S. Li, A. Arisco, and D. F. Balkovetz. Extracellular signal-regulated kinases 1/2 control claudin-2 expression in Madin-Darby canine kidney strain I and II cells. J Biol Chem 2005. 280:3780-8. [72]. X. Hu, Z. Tang, Y. Li, W. Liu, S. Zhang, B. Wang, et al. Deletion of the tyrosine phosphatase Shp2 in Sertoli cells causes infertility in mice. Sci Rep 2015. 5:12982. [73]. M. Cooke, U. Orlando, P. Maloberti, E. J. Podesta, and F. Cornejo Maciel. Tyrosine phosphatase SHP2 regulates the expression of acyl-CoA synthetase ACSL4. J Lipid Res 2011. 52:1936-48. [74]. A. Duarte, A. F. Castillo, R. Castilla, P. Maloberti, C. Paz, E. J. Podesta, et al. An arachidonic acid generation/export system involved in the regulation of cholesterol transport in mitochondria of steroidogenic cells. FEBS Lett 2007. 581:4023-8. [75]. A. Duarte, C. Poderoso, M. Cooke, G. Soria, F. Cornejo Maciel, V. Gottifredi, et al. Mitochondrial fusion is essential for steroid biosynthesis. PLoS One 2012. 7:e45829. [76]. H. Morimoto, K. Iwata, N. Ogonuki, K. Inoue, O. Atsuo, M. Kanatsu-Shinohara, et al. ROS are required for mouse spermatogonial stem cell self-renewal. Cell Stem Cell 2013. 12:774-86. [77]. M. Salvi, A. Stringaro, A. M. Brunati, E. Agostinelli, G. Arancia, G. Clari, et al. Tyrosine phosphatase activity in mitochondria: presence of Shp-2 phosphatase in mitochondria. Cell Mol Life Sci 2004. 61:2393404. [78]. A. Arachiche, O. Augereau, M. Decossas, C. Pertuiset, E. Gontier, T. Letellier, et al. Localization of PTP-1B, SHP-2, and Src exclusively in rat brain mitochondria and functional consequences. J Biol Chem 2008. 283:24406-11. [79]. S. Jakob, J. Altschmied, and J. Haendeler. "Shping 2" different cellular localizations - a potential new player in aging processes. Aging (Albany NY) 2009. 1:664-8. [80]. N. Chughtai, S. Schimchowitsch, J. J. Lebrun, and S. Ali. Prolactin induces SHP-2 association with Stat5, nuclear translocation, and binding to the beta-casein gene promoter in mammary cells. J Biol Chem 2002. 277:31107-14. [81]. T. R. Wu, Y. K. Hong, X. D. Wang, M. Y. Ling, A. M. Dragoi, A. S. Chung, et al. SHP-2 is a dualspecificity phosphatase involved in Stat1 dephosphorylation at both tyrosine and serine residues in nuclei. J Biol Chem 2002. 277:47572-80.
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Figure legends
Figure 1: PTPN11 expression in adult rat testis. A) Confocal fluorescence microscopy analysis of PTPN11 (red) and vimentin (green) in mouse testis shows that PTPN11 is expressed in spermatogonia (Sp) and is localized to Sertoli cell nuclei (S) and cytoplasm between germ cells (arrowheads). PTPN11 is also expressed in in Sertoli cells at the apical side of preleptotene spermatocytes (Pl) and spermatogonia consistent with the position of the BTB. PTPN11 staining is also detected around elongated spermatids (ES) consistent with the attachment sites with Sertoli cells. Modified from Puri and Walker [29] with permission. B) GFRA1 positive cells (left, red) and PTPN11 positive cells (right, green) are shown for a whole mount seminiferous tubule immunofluorescence assay. PTPN11 was found to co-localize with GFRA1 in As cells (SSCs, 1) as well as Apr (2) and Aal4 (4) and Aal8 (8) undifferentiated spermatogonia.
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Figure 2: Hypothesized pathways for PTPN11 stimulation of SSC proliferation, self-renewal and differentiation. A) Sertoli and peritubular myoid cell–derived GDNF binds its receptor, GFRA1, and causes tyrosine phosphorylation of the associated RET tyrosine kinase. FGF produced by Sertoli, Leydig, and germ cells, causes phosphorylation of its receptor, FGFR. PTPN11 is recruited to the receptors and is activated. PTPN11 activation promotes the activation of AKT and ERK. Unidentified targets downstream of AKT and ERK regulate SSC proliferation and renewal (outer arrows). AKT and ERK also stimulate the ETV5 transcription factor that induces expression of BCL6B that promotes SSC proliferation and self-renewal. B) PTPN11 blocks activation of STAT3, which is proposed to regulate the activation of a series of transcription factors that are required to express the differentiation-promoting c-kit protein.
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S1084-9521(16)30020-9 http://dx.doi.org/doi:10.1016/j.semcdb.2016.01.020 YSCDB 1931
To appear in:
Seminars in Cell & Developmental Biology
Received date: Revised date: Accepted date:
25-11-2015 15-1-2016 18-1-2016
Please cite this article as: Puri Pawan, Walker William H.The Regulation of Male Fertility by the PTPN11 Tyrosine Phosphatase.Seminars in Cell and Developmental Biology http://dx.doi.org/10.1016/j.semcdb.2016.01.020 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
The Regulation of Male Fertility by the PTPN11 Tyrosine Phosphatase
Pawan Puria 1 and William H. Walker a
a
Center for Research in Reproductive Physiology, Department of Obstetrics, Gynecology and Reproductive Sciences
Magee Womens Research Institute, University of Pittsburgh, Pittsburgh, PA 15261.
1
Present Address: Division of Nephrology, Department of Pediatrics, University of Pittsburgh School of Medicine,
Pittsburgh, PA 15201 USA. Email: [email protected]
Corresponding author: William H. Walker, Ph.D. Department of Obstetrics, Gynecology and Reproductive Sciences Magee Womens Research Institute, University of Pittsburgh 204 Craft Ave, Room B305, Pittsburgh, PA 15261 USA Tel: 412-641-7672 (office) Fax: 412-641-7676 Email: [email protected]
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Graphical Abstract
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Abstract PTPN11 (also known as SHP2) is a ubiquitously expressed non-receptor tyrosine phosphatase that regulates cell survival, proliferation, differentiation, migration and adhesion. Naturally occurring mutations in the PTPN11 gene cause Noonan and LEOPARD syndromes, two genetic disorders that are characterized by a spectrum of defects including male infertility. This review summarizes four cellular and molecular mechanisms by which PTPN11 acts to support male fertility. First, PTPN11 is required for the proliferation and survival of spermatogonial stem cells (SSCs) that are essential to replenish the germ cells that will become sperm. Second, PTPN11 regulation of cellular adhesion functions in Sertoli cells is required to maintain the blood-testis barrier (BTB) that protects meiotic and post-meiotic germ cells. Third, expression of PTPN11 in Sertoli cells is essential to prevent premature differentiation and exhaustion of the SSC population and to maintain the SSC niche. Finally, in Leydig cells, PTPN11 supports mitochondrial fusion and the expression of acyl-CoA synthetase (ACSL4) needed for the production of steroids including testosterone, which is required for fertility.
Abbreviations PTPN11: protein-tyrosine phosphatase non-receptor type 11, SHP2: SH2-containing tyrosine phosphatase 2, SSC: spermatogonial stem cell, ACSL4: acyl-CoA synthetase, GDNF: glial cell-derived neurotrophic factor, bFGF: basic fibroblast growth factor, BTB: blood-testis barrier, FSH: follicle-stimulating hormone, ZBTB16: zinc finger and BTB domain containing 16, PLZF: promyelocytic leukemia zinc finger, CSF-1: colony stimulating factor 1, CXCL12: chemokine (C-X-C motif) ligand 12, STAT3: Signal transducer and activator of transcription 3, SALL4: Spalt-like transcription factor 4, NGN3: Neurogenin-3, SOHLH1/2: Spermatogenesis and oogenesis specific basic helix-loop-helix proteins 1 and 2, ETV5: Ets Variant 5, BCL6B: B-Cell CLL/Lymphoma 6, TER: transepithelial resistance, HGF: hepatic growth factor, FAK: focal adhesion kinase, CSK: C-Src tyrosine kinase, PKA: protein kinase A, STAR: steroidogenic acute regulatory protein, ACOT2: acyl-CoA thioesterase
Keywords Spermatogenesis; stem cell; testis; male fertility; spermatogonia; self-renewal
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1.1 Introduction 1.1.1 Spermatogenesis and supporting cells. Fertility in males is maintained by the process of spermatogenesis that takes place in the seminiferous tubules of the testis and results in the production of more than 100 million sperm daily in men. The seminiferous tubules contain three main cell types; germ cells, epithelial Sertoli cells and peritubular myoid cells. Spermatogonial stem cells (SSC) are the precursors for all germ cells and are considered to be the foundation of this prolific system. SSCs are rare. Approximately 35,000 SSCs are present in a mouse testis comprising about 0.035% of all germ cells [1]. In rodents, stem cell activity is exhibited in a subpopulation of undifferentiated spermatogonia that are present as single cells (As). The As cells can divide to produce separated As cells that retain stem cell activity or they can divide to produce pairs (Apr) and then chains of 4, 8, 16 and 32 cells called Aaligned (Aal) undifferentiated spermatogonia that increasingly lose stem cell activity [2, 3]. The Aal cells proliferate further by mitotic divisions and enter into a differentiation program that results in the formation of preleptotene spermatocytes that undergo meiosis to produce haploid round spermatids that mature and elongate to form spermatozoa. Sertoli cells function to support germ cells at all stages of development and contribute to the SSC niche by secreting glial cell-derived neurotrophic factor (GDNF) and basic fibroblast growth factor (bFGF) that are critical regulators of SSC fate (reviewed in [4]. Specialized Sertoli-Sertoli cell adhesion complexes create the essential bloodtestis-barrier (BTB) that provides a unique environment for meiosis and the development of haploid germ cells. Sertoli cells also provide attachment sites for the developing mitotic, meiotic and postmeiotic germ cells [5]. Peritubular myoid cells lining the exterior of the seminiferous tubules also produce GDNF and contribute to the support of spermatogenesis [6, 7]. Another important cell type present in the interstitium of the testis is the Leydig cell that secretes testosterone, which is essential for the maintenance of spermatogenesis. Macrophages located in the interstitium and intermingled with peritubular myoid cells also have been recently shown to contribute to the SSC niche [8].
1.1.2 PTPN11 structure and function. PTPN11 (protein-tyrosine phosphatase non-receptor type 11) has a classical protein tyrosine phosphatase (PTP) domain and two N-terminal Src homology 2 (SH2) domains [9, 10]. The phosphatase activity of PTPN11 is modulated by a variety of stimuli including hormones, growth factors and cytokines. In the inactive state, access to the PTP domain is blocked by one of the N-terminal SH2 domains [11-13]. After the binding of appropriate ligands to their receptors, auto4
inhibition is relieved due to the recruitment of the SH2 domains to specific phosphotyrosine sites on liganded receptors or receptor-associated adaptor proteins [9, 10]. PTPN11 is an atypical phosphatase because it can have positive or negative regulatory effects on intracellular kinase-mediated signaling pathways [14, 15]. PTPN11 is known to negatively regulate RhoA activity; whereas, PTPN11 can increase or decrease PI3/AKT and JAK/Stat signaling activity [16, 17]. PTPN11 activates the MAPK pathway via well-characterized mechanisms that deactivate upstream inhibitors of Ras. Specifically, activation of PTPN11 results in the dephosphorylation and deactivation of the sprouty protein negative regulator of Ras and the exclusion of p120RasGAP RAS inhibitor from signaling complexes [18, 19]. The PTPN11-mediated modulation of signaling pathways has been shown to regulate cellular proliferation, differentiation, migration and adhesion in numerous cell types. Because of these properties, PTPN11 is a proto-oncogene. Mutations in PTPN1 have been identified in pediatric hematologic malignancies and sold tumors [20-23].
1.1.3 Male infertility in Noonan and LEOPARD syndrome patients having altered PTPN11 activity. A spectrum of PTPN11 gain-of-function or loss-of-function mutations results in Noonan and LEOPARD syndromes in humans, respectively [14, 15]. These syndromes are characterized by short stature, congenital heart and skeleton abnormalities and reproductive defects such as delayed puberty and male infertility [14, 24, 25]. LEOPARD syndrome patients are reported to have abnormal genitalia; however, no information is available describing the causes and mechanisms of infertility in these patients [26]. Men with Noonan syndrome have fertility defects due to cryptorchidism and their testis were reported to have reduced tubular diameter with fewer germ cells and Leydig cells [27]. Noonan syndrome patients have normal testosterone levels but usually have higher follicle-stimulating hormone (FSH) levels [27]. Some Noonan syndrome patients with normal testicular descent have reduced inhibin levels, a possible indicator of compromised Sertoli cell function and maturity [28]. The information derived from Noonan and LEOPARD patients provided the first indication that PTPN11 was required for male fertility.
2.1 Expression and function of PTPN11 in whole testis studies 2.1.1 Expression and localization of PTPN11 in testicular cells. PTPN11 is ubiquitously expressed in somatic cells. Within the testis, immunofluorescence studies revealed that in Sertoli cells, PTPN11 is expressed in the nuclei and cytoplasm of Sertoli cells and appeared to be concentrated in Sertoli 5
cells near the basement membranes of seminiferous tubules in the vicinity of the BTB [29] (Fig. 1A). Expression levels of PTPN11 remain constant in Sertoli cells through all developmental stages from birth to adulthood. In contrast, PTPN11 levels vary dramatically during male germ cell development [29, 30]. PTPN11 is expressed in the most immature germ cells As through Aaligned spermatogonia (Fig. 1B). In these undifferentiated spermatogonia, the phosphatase was detected in the nucleus and cytoplasm as well as at plasma membrane. Detection of PTPN11 at the plasma membrane is consistent with the known interactions of the phosphatase with plasma membrane receptors [31]. Interestingly, the more mature germ cells in the testis are rare examples of cell types that do not express PTPN11. Specifically, differentiated spermatogonia, spermatocytes and spermatids do not show detectable levels of PTPN11 expression. The mechanisms that regulate the differential expression pattern of PTPN11 in undifferentiated vs. differentiated spermatogonia have not been identified.
2.1.2. PTPN11 is required to maintain spermatogenesis. Global deletion of PTPN11 results in embryonic lethality and precludes the examination of PTPN11 function in the mature testis [32]. Therefore, to determine whether PTPN11 is required to maintain spermatogenesis in an adult mouse, a conditional knockout mouse was created by breeding PTPN11 floxed mice to transgenic mice expressing the Cre recombinase fused to a tamoxifen binding ert2 domain and driven by the ubiquitin promoter [33]. The ubiquitous expression pattern the Cre recombinase allowed the deletion of PTPN11 gene in most of the cells of PTPN11floxed ert2 mice that were exposed to tamoxifen. Histological analysis of the adult testis sections showed that 29 days after PTPN11 deletion there was a loss of less mature germ cells, including spermatogonia and spermatocytes [30]. By 43 days after deletion of PTPN11 there was a loss of all germ cells except the most mature elongating spermatids. The remaining elongated spermatids were distributed randomly and in many cases their acrosomes were mis-oriented relative to the Sertoli cells nucleus suggesting that attachment to the Sertoli cell was mis-regulated. Thus far, the direct targets of PTPN11 that are required to maintain proper orientation of elongated spermatids are not known. By 63 days after PTPN11 KO, all germ cells were absent. These studies suggested that knockout of PTPN11 did not directly affect the survival of differentiated spermatogonia and more mature germ cells. However, the lack of PTPN11 appeared to eliminate the undifferentiated spermatogonia that are required to replenish the maturing germ cells [30]. The timing of the progressive loss of germ cells in global PTPN11 KO mice when compared to the length of spermatogenic cycle suggested that there was a defect near the stem cell stage of development. Consistent with this idea, 6
cells expressing the ZBTB16 transcription factor (also known as PLZF), a marker of SSCs and undifferentiated spermatogonia, were not detected in the testis sections after elimination of PTPN11. These results indicated that PTPN11 deletion resulted in the loss of undifferentiated spermatogonia and perhaps SSCs. This initial global PTPN11 KO model showed that PTPN11 is essential for the maintenance of spermatogenesis. However, because PTPN11 was eliminated in all cells, it remained unresolved whether the progressive loss of germ cells was indicative of an intrinsic germ cell requirement for PTPN11 or whether knock out of PTPN11 disrupted the function of cells that contribute to the SSC niche or the hormonal support of spermatogenesis.
3.1. PTPN11 functions in germ cells 3.1.1 PTPN11 is essential to maintain SSCs and undifferentiated spermatogonia. To determine whether PTPN11 expression in germ cells is required to support spermatogenesis, a germ cell specific PTPN11 KO mouse (GCPTPN11KO) was created by mating PTPN11 floxed mice with Vasa Cre mice so that the gene encoding PTPN11 is specifically deleted in gonocytes (prospermatogonia) at embryonic day 15 prior to the formation of SSCs [30]. Histological analysis of testis sections from GCPTPN11KO mice revealed the progressive loss of germ cells in 3 and 4 week-old KO mice and a complete loss of all germ cells by 8 weeks of age. These results provided unambiguous evidence that PTPN11 is required intrinsically in post-natal germ cells to maintain spermatogenesis. PTPN11 does not appear to be essential in gonocytes because the numbers of these cells were not affected in GCPTPN11KO mice [30]. Two populations of gonocytes are known to exist during the first 5 days after birth in mice [34, 35]. One population gives rise to SSCs that are responsible for all the subsequent waves of spermatogenesis throughout life. A second population of gonocytes is thought to be responsible for a one-time bypassing of the SSC and undifferentiated spermatogonia stages of development. These cells differentiate directly into type A1 differentiated spermatogonia resulting in the initial production of germ cells and sperm called the first wave of spermatogenesis. In agreement with the finding that PTPN11 expression is not detected after the undifferentiated spermatogonia stage of germ cell development, the first wave of spermatogenesis occurred normally in the GCPTPN11KO mice. These results indicated that PTPN11 is not essential for maintaining A1 differentiated spermatogonia and later developmental stages of germ cells. In contrast, SSCs and undifferentiated spermatogonia in GCPTPN11KO mice displayed a 50% decrease in proliferation 5 days after birth and a 60% reduction in the number of these cells by 7 days after birth. Increased rates of apoptosis may account for the removal of the remaining SSCs [30]. These findings provided evidence that PTPN11 is 7
essential for the proliferation of SSCs and thus the perpetuation of spermatogenesis. Similar results were observed using cultures of SSCs, as proliferation was decreased after treatment with a PTPN11 inhibitor. Inhibition of PTPN11 in SSC cultures also decreased the activation of ERK that was mediated by the SSC proliferation promoters bFGF and GDNF [30]. Previously studied conditional knockout mouse models revealed that deletion of PTPN11 compromises the proliferation of numerous cell types including neural stem cells, hematopoietic stem cells (HSCs), cardiac progenitors and T cells. In all of these cell types, PTPN11-mediated activation of ERK activation was shown to be a key and common signaling event [36-38].
3.1.2 PTPN11 and SSC renewal. PTPN11 contributes to the renewal of hematopoietic and neural stem cells [36, 39, 40]. It is not yet known whether PTPN11 contributes to maintaining the stem cell qualities (stemness) of SSCs. Transplantation studies are presently the most reliable measure of stem cell activity [41]. However, traditional transplantation studies in which testes lacking germ cells would be the recipients of PTPN11 deficient SSCs are not likely to be informative because PTPN11 is required for the proliferation and expansion of any colonies that would be derived from the transplanted stem cells. In contrast, transplantation assays in which SSCs containing constitutively active PTPN11 are transplanted could determine that PTPN11 expression increases stem cell activity if the number of colonies resulting from transplantation increases (a result of having more cells with stem cell activity). Similarly, an observation that the number of undifferentiated spermatogonia are increased in knock in mouse models harboring gain of function PTPN11 mutations in germ cells would support the hypothesis that PTPN11 contributes to stem cell activity. Naturally occurring mutations in men have provided information regarding the regulation of SSC self-renewal by PTPN11 [42]. In the testes of men in which sporadic PTPN11 gain of function mutations occur during early germ cell development, there was a preferred clonal expansion of SSCs containing the mutation over genotypically normal SSCs [42]. These findings suggest that a mutation that confers increased PTPN11 activity favors SSC self-renewal and expansion by either increasing the proliferation of SSCs or dampening their differentiation.
3.1.3 Defined and potential PTPN11 contributions to signaling pathways in SSCs that regulate self-renewal and differentiation. The self-renewal of SSCs is promoted by the cytokines GDNF, bFGF, colony stimulating factor 1 (CSF-1), and 8
chemokine (C-X-C motif) ligand 12) (CXCL12) [4]. Although the signaling pathways by which these cytokines support self-renewal of SSCs are not well characterized, it is known that GDNF and bFGF activate the AKT and ERK signaling pathways in cultures enriched for SSCs [30, 43-47]. PTPN11 is a known conduit for GDNF- and bFGF-mediated signaling in other cell types [11, 12, 48-52] and PTPN11 activity is required for GDNF or bFGF-mediated induction of ERK phosphorylation in SSC cultures [30] (Fig. 2A). Thus far, it is unknown whether PTPN11 mediates AKT activation by GDNF and/or bFGF in SSCs. Further studies will be required to determine whether PTPN11 contributes to differentially activating the AKT and ERK signaling pathways as a mechanism to modulate SSCs decisions to self-renew versus differentiate. One potential target for PTPN11-mediated regulation of SSC differentiation is a signaling pathway initiated by the regulation of STAT3, a transcription factor known to promote differentiation and inhibit the self-renewal of SSCs [53] (Fig. 2B). PTPN11 is known to directly dephosphorylate and inactivate STAT3 [54-56]. When active, STAT3 stimulates the expression of SALL4 [57] that in turn can sequester ZBTB16 to allow the expression of the transmembrane receptor cKIT that promotes the differentiation of spermatogonia [58]. STAT3 also induces expression of the NGN3 transcription factor that is proposed to interact with E-Box transcription factors (e.g., SOHOH1/2) to drive the expression of genes promoting differentiation [59]. Thus, PTPN11-mediated down-regulation of STAT3 and NGN3 are candidate mechanisms to block the differentiation of SSCs. Other transcription factors that could be downstream targets of PTPN11 in SSCs are ETV5 and BCL6B that are required for SSC self-renewal [60]. These transcription factors are both up-regulated by bFGF and GDNF via the PTPN11-dependent MAP kinase pathway. Notably, expression of ETV5 is severely down regulated after knockout of PTPN11 in the embryonic kidney, plus knockout of ETV5 or PTPN11 produce similar phenotypes such that no germ cells are produced after the first wave [30, 61]
4.1. PTPN11 functions in Sertoli cells 4.1.1 PTPN11 regulates the integrity of the BTB and attachments of SSCs to their niche. The BTB is a specialized junctional complex composed of tight junctions, adherens junctions and gap junctions between adjacent Sertoli cells that physically divides the seminiferous tubules into basal and apical compartments. Disruption of the BTB causes germ cell differentiation and development to be arrested [62]. The BTB undergoes periodic restructuring to allow passage of spermatocytes from the basal to the adluminal compartment of the testis. Kinasemediated signaling cascades are important for maintaining and remodeling the BTB [63]. Changes in the phosphorylation 9
of cell adhesion proteins are known to regulate their localization at sites of adherens and tight junctions [64]. Expression of a constitutively active PTPN11 mutant (SHP2Q79R) disrupted Sertoli-Sertoli cell adhesion in culture as shown by the decrease in transepithelial resistance (TER), a measure of Sertoli cell tight junction and adherens junction integrity [30]. Hepatic growth factor (HGF), a known regulator of BTB function [65] also decreased TER levels and appears to be an upstream regulator of PTPN11 because HGF stimulation of cultured Sertoli cells increased PTPN11-dependent Src and ERK activity. PTPN11 interactions with an HGF receptor (c-Met) adaptor protein (Gab1) previously were shown to be required to mitigate activation of the MAPK pathway [66]. Importantly, altered Src or ERK activity is known to destabilize adherens and tight junctions [63, 67-71]. Studies using cultured rat Sertoli cells suggest that constitutively active PTPN11 acts by decreasing the phosphorylation of paxillin resulting in the dissociation of the negative regulator Csk from Src followed by ERK activation. Furthermore, focal adhesion kinase (FAK), a mediator of cell-cell adhesion, was inactivated and the actin cytoskeleton, required to stabilize cell-cell adhesion, was disrupted in Sertoli cells expressing SHP2Q79R [29]. Finally, the constitutively active PTPN11 mutant caused the mis-localization of the tight junction protein ZO-1, the adapter protein
-catenin and the transmembrane protein N-cadherin away from cell adhesion
sites at the plasma membrane and into the Sertoli cell cytoplasm [29]. PTPN11 also may contribute to anchoring SSCs within their niche adjacent to Sertoli cells. Inhibition of PTPN11 activity in cultures of SSCs resulted in the dispersal of the three dimensional clumps of SSC colonies and their detachment from feeder cells [30]. This result suggests that PTPN11 is required for attachments between the cells within the SSC colonies and between the SSCs and supporting cells. The disruption of cell attachments in the absence of PTPN11 activity in SSCs or Sertoli cells may contribute to the inability to support SSC proliferation and survival in mice lacking PTPN11.
4.1.2 PTPN11 activity in Sertoli cells is required to maintain SSC self-renewal. Additional information regarding PTPN11 functions in Sertoli cells was generated from a transgenic mouse model in which PTPN11 expression was eliminated specifically in Sertoli cells [72]. This model showed that PTPN11 deficiency caused sterility due to an imbalance in the differentiation and self-renewal of SSCs, a disfunctional BTB and the lack of sperm production. In the absence of PTPN11 in Sertoli cells, seminiferous tubules lacked all germ cells and there was Leydig cell hyperplasia but Sertoli cell numbers were not altered. In the PTPN11 knock out mice, the expression of mRNAs encoding junctional proteins associated with the BTB were altered and a representative group of junctional proteins were found to be mis-localized away from the Sertoli cell plasma membrane resulting in a permeable 10
BTB. As found previously [30], expression of a constitutively active PTPN11 mutant in Sertoli cells also impaired tight junction formation between cultured Sertoli cells [72]. The disruption of the BTB is one likely explanation for the loss of meiotic and post-meiotic germ cells in the Sertoli cell-specific PTPN11 knock out mice. Whereas knock out of PTPN11 in germ cells permanently blocked spermatogenesis by inhibiting the proliferation of SSCs, knock out of PTPN11 in Sertoli cells appears to inappropriately promote the differentiation of SSCs [72]. Specifically, the lack of PTPN11 expression in Sertoli cells resulted in rapidly decreasing numbers of SSC-derived undifferentiated spermatogonia beginning two weeks after birth and the premature appearance of cells having markers of differentiated spermatogonia. Also, the expression of genes encoding differentiation-promoting factors was up-regulated in PTPN11 deficient Sertoli cells although it is not yet clear whether the expression of genes promoting SSC renewal are altered by PTPN11 knock out. Together, these findings suggest that the lack of PTPN11 in Sertoli cells halts the replenishment of germ cells by inducing the differentiation of SSCs that results in the depletion of the SSC population. The requirement for PTPN11 to support SSC renewal was confirmed by the finding that cultures enriched for SSCs and undifferentiated spermatogonia could not be supported by Sertoli cells either lacking PTPN11 or expressing a constitutively active PTPN11. These results indicate that “normal” levels of PTPN11 activity in Sertoli cells are required to maintain the balance of renewal and differentiation of SSCs that in turn are essential for the continuation of spermatogenesis and fertility.
5.1 PTPN11 regulates steroidogenesis in Leydig cells. Leydig cells in the testis interstitium initiate testosterone synthesis when activated by luteinizing hormone (LH) secreted from the pituitary. LH stimulation of Leydig cells increases cAMP levels that activates protein kinase A (PKA) and stimulates a rate-limiting step of steroid synthesis pathway, the transport of cholesterol into the mitochondria, by the up-regulation of a transporter, steroidogenic acute regulatory protein (STAR). The expression of STAR is increased by the production of lipoxygenated or epoxygeneted metabolites of aracadonic acid that are regulated by acyl-CoA synethease (ACSL4) and acyl-CoA thioesterase (ACOT2) [73, 74]. A recent study showed that PTPN11 is a critical regulator of the expression levels of ACSL4 in Leydig cells [73]. Treatment of primary Leydig cell cultures with the PTPN11 inhibitor NSC-87877 decreased cAMP-stimulated testosterone production. Furthermore, decreased steroid production after shRNA-mediated PTPN11 knock down is associated with decreased ACSL4 and StAR protein levels; whereas, PTPN11 overexpression increased ACSL4 and STAR protein levels and steroid production. In addition, mitochondrial fusion, a 11
cAMP-stimulated process that is essential for steroid production was inhibited by shRNA-mediated knockdown of PTPN11 [75]. Together, these data show PTPN11 activation is a key step in the activation of cAMP-PKA pathway stimulated steroid production. Further studies are required to determine whether deletion of PTPN11 specifically in Leydig cells impairs testosterone production and/or male fertility.
6.1 Summary and perspectives 6.1.1 Summary PTPN11 is a critical mediator of functions that are required to maintain spermatogenesis and male fertility. In Sertoli cells, PTPN11 is essential to support the SSC niche and germ cell development. Specifically, PTPN11 expression is required to limit the expression of factors that promote the differentiation of SSCs. Correct levels of PTPN11 activity in Sertoli cells also are required to maintain the BTB and the attachment of SSCs and more mature germ cells. In addition, there is evidence that PTPN11 activity is required in Leydig cells to support the production of steroids including testosterone. Finally, PTPN11 expression in SSCs is essential to maintain the stem cell population that replenishes the germ cells that will become sperm. In the future, defining the signaling pathways regulated by PTPN11 in SSCs could provide important information needed to elucidate the mechanisms by which SSCs determine whether they will selfrenew or differentiate.
6.2.1 Potential regulatory mechanisms of PTPN11 expression and targets in SSCs. Although PTPN11 was found to be required in undifferentiated spermatogoia and Sertoli cells to maintain spermatogenesis and fertility, the molecular mechanisms by which PTPN11 expression and activity are regulated require further examination. For example, PTPN11 is expressed in SSCs and undifferentiated spermatogonia, but PTPN11 cannot be detected in differentiated spermatogonia and more mature germ cells. What is the mechanism by which PTPN11 expression is uniquely shut off in the more differentiated spermatogonia? Does the loss of PTPN11 contribute to the differentiation of spermatogonia? Could this shut off mechanism be useful to slow the progression of tumors that are dependent upon PTPN11? The expression of factors that promote the self-renewal of SSCs including GFRA1 and ZBTB16 decrease in tandem with PTPN11 as the spermatogonia differentiate. Does PTPN11 support the expression of proteins that promote self-renewal as well as mediate intracellular signals associated with self-renewal?
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6.3.1 PTPN11 regulation of oxidative phosphorylation and ROS as a putative mechanism for maintaining SSC selfrenewal Self-renewal and proliferation of SSCs is promoted by reactive oxygen species (ROS) that are generated via activation of ERK and AKT after stimulation of the GFRA1, FGFR receptors [76]. Might PTPN11 activation of ERK and AKT contribute to the generation of ROS resulting in increased proliferation and promoting the self-renewal of SSCs? In other cell types, PTPN11 has been localized to the mitochondria [77, 78] raising the possibility that PTPN11 may act directly to alter oxidative phosphorylation and ROS production in SSCs and Sertoli cells. In fact, normal levels of PTPN11 activity in the mitochondria are proposed to keep ROS levels lower in the mitochondria, decrease DNA damage and slow the aging process [79], which are properties that would promote the fidelity and longevity of SSCs. One hypothesis that would unify the information available regarding PTPN11 and ROS production is that in SSCs, PTPN11 in the mitochondria acts to limit ROS production and inhibits aging, but when signaling through the GFRA1 and FGF receptors increases PTPN11 in the cytoplasm proliferation promoting signaling pathways are activated resulting in temporarily elevated ROS production.
6.4.1 Transcriptional regulation by PTPN11 may promote self-renewal PTPN11 has also been localized to the nucleus and acts to regulate transcription by interacting directly with the DNA bound signal transducer and activator of transcription 5 (STAT5) or by dephosphorylating and inactivating STAT1. [80, 81]. Interestingly PTPN11 also has been shown to directly dephosphorylate and inactivate STAT3 [55, 56], which in its active state enhances the expression of factors that promote the differentiation of SSCs [57, 59]. Additional studies will be required to determine whether PTPN11 mediates the inactivation of STAT3 in SSCs and acts to support their selfrenewal.
6.5.1 Future benefits from studies of PTPN11 in the testes The specific mechanisms by which PTPN11 support male fertility remain to be fully revealed. However, because it was found that PTPN11 activity is essential to maintain spermatogenesis, at least one practical application can be envisioned from the advances in understanding the functions of PTPN11. Specifically, inhibitors of PTPN11 activity delivered to the testes could be used to eliminate SSCs and permanently halt sperm production. In fact, an inhibitor of PTPN11 was found to eliminate SSCs and more mature germ cells in mouse testes [30], The development of a sterilant 13
strategy using PTPN11 inhibitors could be used to control animal populations, replace neutering surgeries or used as a replacement for vasectomies. Other possible contributions from PTPN11 studies might be improved strategies to more efficiently culture SSCs for further research or for transplantation into germ cell deficient testes to restore fertility after chemotherapy. Finally, a better understanding of the factors regulated by PTPN11 may facilitate the production of therapies for infertility conditions caused by disruption of SHP2 regulated pathways.
Acknowledgement We thank Dr. Anthony Zeleznik for assistance in the editing of the manuscript.
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References
[1]. R. A. Tegelenbosch, and D. G. de Rooij. A quantitative study of spermatogonial multiplication and stem cell renewal in the C3H/101 F1 hybrid mouse. Mutat Res 1993. 290:193-200. [2]. T. Nakagawa, M. Sharma, Y. Nabeshima, R. E. Braun, and S. Yoshida. Functional hierarchy and reversibility within the murine spermatogenic stem cell compartment. Science 2010. 328:62-7. [3]. K. Hara, T. Nakagawa, H. Enomoto, M. Suzuki, M. Yamamoto, B. D. Simons, et al. Mouse spermatogenic stem cells continually interconvert between equipotent singly isolated and syncytial states. Cell Stem Cell 2014. 14:658-72. [4]. S. R. Chen, and Y. X. Liu. Regulation of spermatogonial stem cell self-renewal and spermatocyte meiosis by Sertoli cell signaling. Reproduction 2015. 149:R159-67. [5]. C. Y. Cheng, and D. D. Mruk. The blood-testis barrier and its implications for male contraception. Pharmacol Rev 2012. 64:16-64. [6]. L. Y. Chen, P. R. Brown, W. B. Willis, and E. M. Eddy. Peritubular myoid cells participate in male mouse spermatogonial stem cell maintenance. Endocrinology 2014. 155:4964-74. [7]. M. Welsh, L. Moffat, L. Jack, A. McNeilly, D. Brownstein, P. T. Saunders, et al. Deletion of androgen receptor in the smooth muscle of the seminal vesicles impairs secretory function and alters its responsiveness to exogenous testosterone and estradiol. Endocrinology 2010. 151:3374-85. [8]. T. DeFalco, S. J. Potter, A. V. Williams, B. Waller, M. J. Kan, and B. Capel. Macrophages Contribute to the Spermatogonial Niche in the Adult Testis. Cell Rep 2015. 12:1107-19. [9]. D. Barford, and B. G. Neel. Revealing mechanisms for SH2 domain mediated regulation of the protein tyrosine phosphatase SHP-2. Structure 1998. 6:249-54. [10]. P. Hof, S. Pluskey, S. Dhe-Paganon, M. J. Eck, and S. E. Shoelson. Crystal structure of the tyrosine phosphatase SHP-2. Cell 1998. 92:441-50. [11]. M. Perrinjaquet, M. Vilar, and C. F. Ibanez. Protein-tyrosine phosphatase SHP2 contributes to GDNF neurotrophic activity through direct binding to phospho-Tyr687 in the RET receptor tyrosine kinase. J Biol Chem 2010. 285:31867-75. [12]. H. Li, C. Tao, Z. Cai, K. Hertzler-Schaefer, T. N. Collins, F. Wang, et al. Frs2alpha and Shp2 signal independently of Gab to mediate FGF signaling in lens development. J Cell Sci 2014. 127:571-82. [13]. J. G. Bode, J. Schweigart, J. Kehrmann, C. Ehlting, F. Schaper, P. C. Heinrich, et al. TNF-alpha induces tyrosine phosphorylation and recruitment of the Src homology protein-tyrosine phosphatase 2 to the gp130 signal-transducing subunit of the IL-6 receptor complex. J Immunol 2003. 171:257-66. [14]. M. Tajan, A. de Rocca Serra, P. Valet, T. Edouard, and A. Yart. SHP2 sails from physiology to pathology. Eur J Med Genet 2015. 58:509-25. [15]. K. S. Grossmann, M. Rosario, C. Birchmeier, and W. Birchmeier. The tyrosine phosphatase Shp2 in development and cancer. Adv Cancer Res 2010. 106:53-89. [16]. S. Q. Zhang, W. G. Tsiaras, T. Araki, G. Wen, L. Minichiello, R. Klein, et al. Receptor-specific regulation of phosphatidylinositol 3'-kinase activation by the protein tyrosine phosphatase Shp2. Mol Cell Biol 2002. 22:4062-72. [17]. Y. Ke, J. Lesperance, E. E. Zhang, E. A. Bard-Chapeau, R. G. Oshima, W. J. Muller, et al. Conditional deletion of Shp2 in the mammary gland leads to impaired lobulo-alveolar outgrowth and attenuated Stat5 activation. J Biol Chem 2006. 281:34374-80. [18]. Y. M. Agazie, and M. J. Hayman. Molecular mechanism for a role of SHP2 in epidermal growth factor receptor signaling. Mol Cell Biol 2003. 23:7875-86. [19]. H. Hanafusa, S. Torii, T. Yasunaga, K. Matsumoto, and E. Nishida. Shp2, an SH2-containing proteintyrosine phosphatase, positively regulates receptor tyrosine kinase signaling by dephosphorylating and inactivating the inhibitor Sprouty. J Biol Chem 2004. 279:22992-5. [20]. R. J. Chan, and G. S. Feng. PTPN11 is the first identified proto-oncogene that encodes a tyrosine phosphatase. Blood 2007. 109:862-7.
15
[21]. M. Tartaglia, C. M. Niemeyer, A. Fragale, X. Song, J. Buechner, A. Jung, et al. Somatic mutations in PTPN11 in juvenile myelomonocytic leukemia, myelodysplastic syndromes and acute myeloid leukemia. Nat Genet 2003. 34:148-50. [22]. M. L. Loh, S. Vattikuti, S. Schubbert, M. G. Reynolds, E. Carlson, K. H. Lieuw, et al. Mutations in PTPN11 implicate the SHP-2 phosphatase in leukemogenesis. Blood 2004. 103:2325-31. [23]. M. Bentires-Alj, J. G. Paez, F. S. David, H. Keilhack, B. Halmos, K. Naoki, et al. Activating mutations of the noonan syndrome-associated SHP2/PTPN11 gene in human solid tumors and adult acute myelogenous leukemia. Cancer Res 2004. 64:8816-20. [24]. A. A. Jorge, A. C. Malaquias, I. J. Arnhold, and B. B. Mendonca. Noonan syndrome and related disorders: a review of clinical features and mutations in genes of the RAS/MAPK pathway. Horm Res 2009. 71:185-93. [25]. I. van der Burgt, E. Berends, E. Lommen, S. van Beersum, B. Hamel, and E. Mariman. Clinical and molecular studies in a large Dutch family with Noonan syndrome. Am J Med Genet 1994. 53:187-91. [26]. E. Nemes, K. Farkas, B. Kocsis-Deak, A. Drubi, A. Sulak, K. Tripolszki, et al. Phenotypical diversity of patients with LEOPARD syndrome carrying the worldwide recurrent p.Tyr279Cys PTPN11 mutation. Arch Dermatol Res 2015. 307:891-5. [27]. I. Sasagawa, T. Nakada, Y. Kubota, T. Sawamura, T. Tateno, and M. Ishigooka. Gonadal function and testicular histology in Noonan's syndrome with bilateral cryptorchidism. Arch Androl 1994. 32:135-40. [28]. K. A. Marcus, C. G. Sweep, I. van der Burgt, and C. Noordam. Impaired Sertoli cell function in males diagnosed with Noonan syndrome. J Pediatr Endocrinol Metab 2008. 21:1079-84. [29]. P. Puri, and W. H. Walker. The tyrosine phosphatase SHP2 regulates Sertoli cell junction complexes. Biol Reprod 2013. 88:59. [30]. P. Puri, B. T. Phillips, H. Suzuki, K. E. Orwig, A. Rajkovic, P. E. Lapinski, et al. The transition from stem cell to progenitor spermatogonia and male fertility requires the SHP2 protein tyrosine phosphatase. Stem Cells 2014. 32:741-53. [31]. Y. Fujioka, T. Matozaki, T. Noguchi, A. Iwamatsu, T. Yamao, N. Takahashi, et al. A novel membrane glycoprotein, SHPS-1, that binds the SH2-domain-containing protein tyrosine phosphatase SHP-2 in response to mitogens and cell adhesion. Mol Cell Biol 1996. 16:6887-99. [32]. T. M. Saxton, M. Henkemeyer, S. Gasca, R. Shen, D. J. Rossi, F. Shalaby, et al. Abnormal mesoderm patterning in mouse embryos mutant for the SH2 tyrosine phosphatase Shp-2. EMBO J 1997. 16:2352-64. [33]. T. J. Bauler, N. Kamiya, P. E. Lapinski, E. Langewisch, Y. Mishina, J. E. Wilkinson, et al. Development of severe skeletal defects in induced SHP-2-deficient adult mice: a model of skeletal malformation in humans with SHP-2 mutations. Dis Model Mech 2011. 4:228-39. [34]. P. M. Kluin, and D. G. de Rooij. A comparison between the morphology and cell kinetics of gonocytes and adult type undifferentiated spermatogonia in the mouse. Int J Androl 1981. 4:475-93. [35]. S. Yoshida, M. Sukeno, T. Nakagawa, K. Ohbo, G. Nagamatsu, T. Suda, et al. The first round of mouse spermatogenesis is a distinctive program that lacks the self-renewing spermatogonia stage. Development 2006. 133:1495-505. [36]. G. Chan, L. S. Cheung, W. Yang, M. Milyavsky, A. D. Sanders, S. Gu, et al. Essential role for Ptpn11 in survival of hematopoietic stem and progenitor cells. Blood 2011. 117:4253-61. [37]. Y. G. Langdon, S. C. Goetz, A. E. Berg, J. T. Swanik, and F. L. Conlon. SHP-2 is required for the maintenance of cardiac progenitors. Development 2007. 134:4119-30. [38]. T. V. Nguyen, Y. Ke, E. E. Zhang, and G. S. Feng. Conditional deletion of Shp2 tyrosine phosphatase in thymocytes suppresses both pre-TCR and TCR signals. J Immunol 2006. 177:5990-6. [39]. H. H. Zhu, K. Ji, N. Alderson, Z. He, S. Li, W. Liu, et al. Kit-Shp2-Kit signaling acts to maintain a functional hematopoietic stem and progenitor cell pool. Blood 2011. 117:5350-61. [40]. Y. Ke, E. E. Zhang, K. Hagihara, D. Wu, Y. Pang, R. Klein, et al. Deletion of Shp2 in the brain leads to defective proliferation and differentiation in neural stem cells and early postnatal lethality. Mol Cell Biol 2007. 27:6706-17. [41]. J. M. Oatley, and R. L. Brinster. Spermatogonial stem cells. Methods Enzymol 2006. 419:259-82. 16
[42]. S. R. Yoon, S. K. Choi, J. Eboreime, B. D. Gelb, P. Calabrese, and N. Arnheim. Age-dependent germline mosaicism of the most common noonan syndrome mutation shows the signature of germline selection. Am J Hum Genet 2013. 92:917-26. [43]. L. Braydich-Stolle, N. Kostereva, M. Dym, and M. C. Hofmann. Role of Src family kinases and N-Myc in spermatogonial stem cell proliferation. Dev Biol 2007. 304:34-45. [44]. J. M. Oatley, M. R. Avarbock, and R. L. Brinster. Glial cell line-derived neurotrophic factor regulation of genes essential for self-renewal of mouse spermatogonial stem cells is dependent on Src family kinase signaling. J Biol Chem 2007. 282:25842-51. [45]. J. Lee, M. Kanatsu-Shinohara, K. Inoue, N. Ogonuki, H. Miki, S. Toyokuni, et al. Akt mediates selfrenewal division of mouse spermatogonial stem cells. Development 2007. 134:1853-9. [46]. Z. He, J. Jiang, M. Kokkinaki, N. Golestaneh, M. C. Hofmann, and M. Dym. Gdnf upregulates c-Fos transcription via the Ras/Erk1/2 pathway to promote mouse spermatogonial stem cell proliferation. Stem Cells 2008. 26:266-78. [47]. K. Ishii, M. Kanatsu-Shinohara, S. Toyokuni, and T. Shinohara. FGF2 mediates mouse spermatogonial stem cell self-renewal via upregulation of Etv5 and Bcl6b through MAP2K1 activation. Development 2012. 139:1734-43. [48]. R. Willecke, J. Heuberger, K. Grossmann, O. Michos, K. Schmidt-Ott, K. Walentin, et al. The tyrosine phosphatase Shp2 acts downstream of GDNF/Ret in branching morphogenesis of the developing mouse kidney. Dev Biol 2011. 360:310-7. [49]. A. D'Alessio, D. Califano, M. Incoronato, G. Santelli, T. Florio, G. Schettini, et al. The tyrosine phosphatase Shp-2 mediates intracellular signaling initiated by Ret mutants. Endocrinology 2003. 144:4298305. [50]. Z. Ahmed, C. C. Lin, K. M. Suen, F. A. Melo, J. A. Levitt, K. Suhling, et al. Grb2 controls phosphorylation of FGFR2 by inhibiting receptor kinase and Shp2 phosphatase activity. J Cell Biol 2013. 200:493-504. [51]. Y. Pan, C. Carbe, A. Powers, E. E. Zhang, J. D. Esko, K. Grobe, et al. Bud specific N-sulfation of heparan sulfate regulates Shp2-dependent FGF signaling during lacrimal gland induction. Development 2008. 135:301-10. [52]. J. Burks, and Y. M. Agazie. Modulation of alpha-catenin Tyr phosphorylation by SHP2 positively effects cell transformation induced by the constitutively active FGFR3. Oncogene 2006. 25:7166-79. [53]. J. M. Oatley, A. V. Kaucher, M. R. Avarbock, and R. L. Brinster. Regulation of mouse spermatogonial stem cell differentiation by STAT3 signaling. Biol Reprod 2010. 83:427-33. [54]. E. E. Zhang, E. Chapeau, K. Hagihara, and G. S. Feng. Neuronal Shp2 tyrosine phosphatase controls energy balance and metabolism. Proc Natl Acad Sci U S A 2004. 101:16064-9. [55]. Y. Yang, B. Jiang, Y. Huo, L. Primo, J. S. Dahl, T. L. Benjamin, et al. Shp2 suppresses PyMT-induced transformation in mouse fibroblasts by inhibiting Stat3 activity. Virology 2011. 409:204-10. [56]. D. J. Kim, M. L. Tremblay, and J. Digiovanni. Protein tyrosine phosphatases, TC-PTP, SHP1, and SHP2, cooperate in rapid dephosphorylation of Stat3 in keratinocytes following UVB irradiation. PLoS One 2010. 5:e10290. [57]. J. D. Bard, P. Gelebart, H. M. Amin, L. C. Young, Y. Ma, and R. Lai. Signal transducer and activator of transcription 3 is a transcriptional factor regulating the gene expression of SALL4. FASEB J 2009. 23:140514. [58]. R. M. Hobbs, S. Fagoonee, A. Papa, K. Webster, F. Altruda, R. Nishinakamura, et al. Functional antagonism between Sall4 and Plzf defines germline progenitors. Cell Stem Cell 2012. 10:284-98. [59]. A. V. Kaucher, M. J. Oatley, and J. M. Oatley. NEUROG3 is a critical downstream effector for STAT3regulated differentiation of mammalian stem and progenitor spermatogonia. Biol Reprod 2012. 86:164, 1-11. [60]. J. A. Schmidt, M. R. Avarbock, J. W. Tobias, and R. L. Brinster. Identification of glial cell line-derived neurotrophic factor-regulated genes important for spermatogonial stem cell self-renewal in the rat. Biol Reprod 2009. 81:56-66.
17
[61]. G. Tyagi, K. Carnes, C. Morrow, N. V. Kostereva, G. C. Ekman, D. D. Meling, et al. Loss of Etv5 decreases proliferation and RET levels in neonatal mouse testicular germ cells and causes an abnormal first wave of spermatogenesis. Biol Reprod 2009. 81:258-66. [62]. C. H. Wong, and C. Y. Cheng. The blood-testis barrier: its biology, regulation, and physiological role in spermatogenesis. Curr Top Dev Biol 2005. 71:263-96. [63]. C. H. Wong, and C. Y. Cheng. Mitogen-activated protein kinases, adherens junction dynamics, and spermatogenesis: a review of recent data. Dev Biol 2005. 286:1-15. [64]. J. C. Li, D. Mruk, and C. Y. Cheng. The inter-Sertoli tight junction permeability barrier is regulated by the interplay of protein phosphatases and kinases: an in vitro study. J Androl 2001. 22:847-56. [65]. A. Catizone, G. Ricci, M. Caruso, F. Ferranti, R. Canipari, and M. Galdieri. Hepatocyte growth factor (HGF) regulates blood-testis barrier (BTB) in adult rats. Mol Cell Endocrinol 2012. 348:135-146. [66]. U. Schaeper, N. H. Gehring, K. P. Fuchs, M. Sachs, B. Kempkes, and W. Birchmeier. Coupling of Gab1 to c-Met, Grb2, and Shp2 mediates biological responses. J Cell Biol 2000. 149:1419-32. [67]. R. M. Ray, R. J. Vaidya, and L. R. Johnson. MEK/ERK regulates adherens junctions and migration through Rac1. Cell Motil Cytoskeleton 2007. 64:143-56. [68]. S. A. Woodcock, C. Rooney, M. Liontos, Y. Connolly, V. Zoumpourlis, A. D. Whetton, et al. SRCinduced disassembly of adherens junctions requires localized phosphorylation and degradation of the rac activator tiam1. Mol Cell 2009. 33:639-53. [69]. N. P. Lee, and C. Y. Cheng. Protein kinases and adherens junction dynamics in the seminiferous epithelium of the rat testis. J Cell Physiol 2005. 202:344-60. [70]. Y. Wang, J. Zhang, X. J. Yi, and F. S. Yu. Activation of ERK1/2 MAP kinase pathway induces tight junction disruption in human corneal epithelial cells. Exp Eye Res 2004. 78:125-36. [71]. J. H. Lipschutz, S. Li, A. Arisco, and D. F. Balkovetz. Extracellular signal-regulated kinases 1/2 control claudin-2 expression in Madin-Darby canine kidney strain I and II cells. J Biol Chem 2005. 280:3780-8. [72]. X. Hu, Z. Tang, Y. Li, W. Liu, S. Zhang, B. Wang, et al. Deletion of the tyrosine phosphatase Shp2 in Sertoli cells causes infertility in mice. Sci Rep 2015. 5:12982. [73]. M. Cooke, U. Orlando, P. Maloberti, E. J. Podesta, and F. Cornejo Maciel. Tyrosine phosphatase SHP2 regulates the expression of acyl-CoA synthetase ACSL4. J Lipid Res 2011. 52:1936-48. [74]. A. Duarte, A. F. Castillo, R. Castilla, P. Maloberti, C. Paz, E. J. Podesta, et al. An arachidonic acid generation/export system involved in the regulation of cholesterol transport in mitochondria of steroidogenic cells. FEBS Lett 2007. 581:4023-8. [75]. A. Duarte, C. Poderoso, M. Cooke, G. Soria, F. Cornejo Maciel, V. Gottifredi, et al. Mitochondrial fusion is essential for steroid biosynthesis. PLoS One 2012. 7:e45829. [76]. H. Morimoto, K. Iwata, N. Ogonuki, K. Inoue, O. Atsuo, M. Kanatsu-Shinohara, et al. ROS are required for mouse spermatogonial stem cell self-renewal. Cell Stem Cell 2013. 12:774-86. [77]. M. Salvi, A. Stringaro, A. M. Brunati, E. Agostinelli, G. Arancia, G. Clari, et al. Tyrosine phosphatase activity in mitochondria: presence of Shp-2 phosphatase in mitochondria. Cell Mol Life Sci 2004. 61:2393404. [78]. A. Arachiche, O. Augereau, M. Decossas, C. Pertuiset, E. Gontier, T. Letellier, et al. Localization of PTP-1B, SHP-2, and Src exclusively in rat brain mitochondria and functional consequences. J Biol Chem 2008. 283:24406-11. [79]. S. Jakob, J. Altschmied, and J. Haendeler. "Shping 2" different cellular localizations - a potential new player in aging processes. Aging (Albany NY) 2009. 1:664-8. [80]. N. Chughtai, S. Schimchowitsch, J. J. Lebrun, and S. Ali. Prolactin induces SHP-2 association with Stat5, nuclear translocation, and binding to the beta-casein gene promoter in mammary cells. J Biol Chem 2002. 277:31107-14. [81]. T. R. Wu, Y. K. Hong, X. D. Wang, M. Y. Ling, A. M. Dragoi, A. S. Chung, et al. SHP-2 is a dualspecificity phosphatase involved in Stat1 dephosphorylation at both tyrosine and serine residues in nuclei. J Biol Chem 2002. 277:47572-80.
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Figure legends
Figure 1: PTPN11 expression in adult rat testis. A) Confocal fluorescence microscopy analysis of PTPN11 (red) and vimentin (green) in mouse testis shows that PTPN11 is expressed in spermatogonia (Sp) and is localized to Sertoli cell nuclei (S) and cytoplasm between germ cells (arrowheads). PTPN11 is also expressed in in Sertoli cells at the apical side of preleptotene spermatocytes (Pl) and spermatogonia consistent with the position of the BTB. PTPN11 staining is also detected around elongated spermatids (ES) consistent with the attachment sites with Sertoli cells. Modified from Puri and Walker [29] with permission. B) GFRA1 positive cells (left, red) and PTPN11 positive cells (right, green) are shown for a whole mount seminiferous tubule immunofluorescence assay. PTPN11 was found to co-localize with GFRA1 in As cells (SSCs, 1) as well as Apr (2) and Aal4 (4) and Aal8 (8) undifferentiated spermatogonia.
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Figure 2: Hypothesized pathways for PTPN11 stimulation of SSC proliferation, self-renewal and differentiation. A) Sertoli and peritubular myoid cell–derived GDNF binds its receptor, GFRA1, and causes tyrosine phosphorylation of the associated RET tyrosine kinase. FGF produced by Sertoli, Leydig, and germ cells, causes phosphorylation of its receptor, FGFR. PTPN11 is recruited to the receptors and is activated. PTPN11 activation promotes the activation of AKT and ERK. Unidentified targets downstream of AKT and ERK regulate SSC proliferation and renewal (outer arrows). AKT and ERK also stimulate the ETV5 transcription factor that induces expression of BCL6B that promotes SSC proliferation and self-renewal. B) PTPN11 blocks activation of STAT3, which is proposed to regulate the activation of a series of transcription factors that are required to express the differentiation-promoting c-kit protein.
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