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Functional analysis of ZFP36 proteins in keratinocytes
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Research paper
Functional analysis of ZFP36 proteins in keratinocytes Frauke Prenzler, Annunziata Fragasso, Angelika Schmitt, Barbara Munz ∗ University Hospital Tübingen, Medical Clinic, Department of Sports Medicine, Hoppe-Seyler-Str. 6, D-72076 Tübingen, Germany
a r t i c l e
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Article history: Received 13 July 2015 Received in revised form 28 April 2016 Accepted 28 April 2016 Keywords: ZFP36 proteins Epidermis Keratinocytes
a b s t r a c t The ZFP36 family of zinc finger proteins, including ZFP36, ZFP36L1, and ZFP36L2, regulates the production of growth factors and cytokines via destabilization of the respective mRNAs. We could recently demonstrate that in cultured keratinocytes, expression of the ZFP36, ZFP36L1, and ZFP36L2 genes is induced by growth factors and cytokines and that ZFP36L1 is a potent regulator of keratinocyte VEGF production. We now further analyzed the localization and function of ZFP36 proteins in the skin, specifically in epidermal keratinocytes. We found that in human epidermis, the ZFP36 protein could be detected in basal and suprabasal keratinocytes, whereas ZFP36L1 and ZFP36L2 were expressed mainly in the basal layer, indicating different and non-redundant functions of the three proteins in the epidermis. Consistently, upon inhibition of ZFP36 or ZFP36L1 expression using specific siRNAs, there was no major effect on expression of the respective other gene. In addition, we demonstrate that both ZFP36 and ZFP36L1 influence keratinocyte cell cycle, differentiation, and apoptosis in a distinct manner. Finally, we show that similarly as ZFP36L1, ZFP36 is a potent regulator of keratinocyte VEGF production. Thus, it is likely that both proteins regulate angiogenesis via paracrine mechanisms. Taken together, our results suggest that ZFP36 proteins might control reepithelialization and angiogenesis in the skin in a multimodal manner. © 2016 Elsevier GmbH. All rights reserved.
1. Introduction Cutaneous wound repair is a highly complex process involving many different cell types, such as keratinocytes, fibroblasts, endothelial cells, and immune cells. The different stages of healing—inflammation, reepithelialization, contraction and angiogenesis—are regulated by a multitude of growth factors and cytokines, such as EGF, TGF, FGF1, KGF, HGF, VEGF, and TNF␣. The epidermis, which forms the outer layer of the skin, consists mainly of keratinocytes. They differentiate from the basal layer, however, during wound healing, epidermal keratinocytes migrate into the wound site and become hyperproliferative (reviewed by Martin, 1997; Gurtner et al., 2008). ZFP36 (tristetraprolin TTP, Tis11, Nup34), ZFP36L1 (Tis11B, ERF1, BRF1, berg36), and ZFP36L2 (Tis11D, ERF2, BRF2) are members of the ZFP36 family of RNA-binding zinc finger proteins (DuBois et al., 1995; Varnum et al., 1991). They appear to be evolutionary “old” proteins, since ZFP36 proteins have been identified in a broad variety of species from yeast (Ma and Herschman, 1995) to
∗ Corresponding author. E-mail address: [email protected] (B. Munz).
humans (Taylor et al., 1991), except for the ZFP36L3 isoform, which seems to be mouse-specific (Blackshear et al., 2005). ZFP36 proteins bind to and destabilize specific target mRNAs, such as the TNF˛, the GM-CSF, and the VEGF transcripts, thus reducing expression of the respective genes. Recognition occurs via binding of the ZFP36 proteins to AU-rich elements (AREs) within the target transcripts’ 3 untranslated regions (3 UTRs) (Lai et al., 2000; Carballo et al., 2000; Ciais et al., 2004; for review, see Baou et al., 2009; Sanduja et al., 2011; Ciais et al., 2013). Despite the fact that AREs were first discovered in cytokine and protooncogene sequences (Caput et al., 1986), genome-wide screens have identified AREs in a broad variety of different mRNAs encoding proteins of multiple and diverse functions (Bakheet et al., 2001; Lai et al., 2006). Expression of the ZFP36/Zfp36 (human/rodent), ZFP36L1/Zfp36l1 and ZFP36L2/Zfp36l2 genes can be induced by various stimuli, e.g. growth factors, in a broad range of cell types (Varnum et al., 1989a,b; Gomperts et al., 1992; Reppe et al., 2004; Manabe et al., 1999). All three genes are expressed in a huge variety of cell types and tissues, however, each has its unique and distinct spatial and temporal expression pattern (Carrick and Blackshear, 2007). With respect to the physiological role of the ZFP36 proteins under in vivo conditions, studies in knock-out mice demonstrated roles
http://dx.doi.org/10.1016/j.ejcb.2016.04.007 0171-9335/© 2016 Elsevier GmbH. All rights reserved.
Please cite this article in press as: Prenzler, F., et al., Functional analysis of ZFP36 proteins in keratinocytes. Eur. J. Cell Biol. (2016), http://dx.doi.org/10.1016/j.ejcb.2016.04.007
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Fig. 1. Localization of ZFP36, ZFP36L1, and ZFP36L2 in the skin. Expression of the three ZFP36 genes was analyzed in sections from human belly skin as indicated. Co-staining was performed with an antibody directed against the proliferation -associated keratin 14. Nuclei were stained with DAPI.
Fig. 2. Expression of ZFP36 and ZFP36L1 after scratch-wounding at the RNA and at the protein level. qPCR and Western blot analysis were performed on RNA and protein samples derived from quiescent, scratch-wounded keratinocyte monolayers as indicated. The blots were also probed with an antibody directed against GAPDH as a loading control and a reference for normalization. Data are expressed as means +/− SD (n = 5). *: p < 0.05; **: p < 0.01.
for ZFP36, ZFP36L1, and ZFP36L2, in inflammation, chorioallantoic fusion and vascularization, and early embryonic development, respectively (Taylor et al., 1996; Stumpo et al., 2004; Ramos et al., 2004; Bell et al., 2006). In addition, ZFP36L1 has been implicated in myogenesis (Busse et al., 2008), and ZFP36 has been identified as regulator of dendritic cell maturation (Emmons et al., 2008), but little is known so far about a possible role of ZFP36 proteins in keratinocytes or in cutaneous wound repair.
We could recently demonstrate induction of all three ZFP36 genes, specifically of ZFP36 and ZFP36L1, after scratch-wounding in vitro. In addition, we could show that ZFP36L1 regulates keratinocyte VEGF production, thereby potentially modulating the angiogenic response in wound tissue (Hacker et al., 2010). Furthermore, most interestingly, a recent report demonstrated that the anti-inflammatory effect of glucocorticoids on the epidermis is in part mediated by ZFP36 (Sevilla et al., 2015).
Please cite this article in press as: Prenzler, F., et al., Functional analysis of ZFP36 proteins in keratinocytes. Eur. J. Cell Biol. (2016), http://dx.doi.org/10.1016/j.ejcb.2016.04.007
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Fig. 3. Inhibition of ZFP36, ZFP36L1, and ZFP36L2 gene expression in cultured keratinocytes. HaCaT keratinocytes were transfected with specific siRNAs directed against ZFP36 or ZFP36L1, an unspecific (“scrambled”) negative control, or left untreated. (A) To assess the efficiency of the transfection procedure, the cells were first transfected with a FITC-labelled “scrambled” siRNA and analyzed by FACS sorting. (B) After inhibition of expression of ZFP36 or ZFP36L1 via siRNA transfection, cells were rendered quiescent by serum starvation and subsequently scratch-wounded. Expression of ZFP36 and ZFP36L1 was analyzed by qPCR. Data are expressed as means +/− SD (n = 2). *: p < 0.05; **: p < 0.01. (C) ZFP36L1 expression was also analyzed by Western blot 24 h after transfection (without scratching). GAPDH levels were assessed as a control for equal loading.
Now, we functionally analyzed the role of the three ZFP36 proteins in epidermal keratinocytes. Specifically, we demonstrate that each of the three genes has its specific and unique spatial expression pattern in the epidermis and that inhibiting ZFP36 or ZFP36L1 expression in cultured keratinocytes induces apoptosis, blocks cell proliferation, and enhances VEGF production. Our data indicate that ZFP36 proteins might be multi-functional players in the skin and perhaps also in wound healing.
2. Experimental procedures 2.1. Skin tissue Paraffin sections from human belly skin were purchased from Zyagen, San Diego, CA, USA.
2.2. Cell culture Human HaCaT keratinocytes were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum.
2.4. Transfection of keratinocytes with siRNA 1 × 106 HaCaT keratinocytes per well were trypsinized, washed, resuspended and reverse-transfected with ZFP36-, or ZFP36L1specific siRNA or an unspecific (“scrambled”, scr) siRNA as a negative control (final concentration of all siRNAs: 100 nM). 8 l Lipofectamine (Life Technologies) were used as a transfection reagent. After incubation in 6-well plates at 37 ◦ C for 24 h, the medium was replaced by regular or serum-free medium. 2.5. RNA isolation and qPCR RNA isolation was performed using the AllPrep kit (Qiagen). Semi-quantitative real time PCR analysis was carried out using the iCycler MyiQ system (Bio-Rad). Gene expression was analyzed using the Eva Green Mastermix (Bio-Rad). For detection of different transcripts, pre-designed primers (Qiagen QuantiTect Primer Assays) were used. In each experiment, melting curve analysis was performed to verify that a single transcript was produced. RT-qPCR relative gene expression was calculated using the comparative CT (2−DC T ) method, where expression was normalized to RPL13 and GAPDH. Non-RT- and non-template controls were run for all reactions.
2.3. Scratch-wounding 2.6. Generation of protein extracts and Western blotting HaCaT cells were grown in 6-well plates and serum-starved overnight. They were then scratched with a scalpel to yield wounds with uniform width.
Whole cell protein lysates were generated as follows: cells were harvested in PBS and cell pellets were resuspended in
Please cite this article in press as: Prenzler, F., et al., Functional analysis of ZFP36 proteins in keratinocytes. Eur. J. Cell Biol. (2016), http://dx.doi.org/10.1016/j.ejcb.2016.04.007
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Fig. 4. Analysis of proliferation, differentiation, and apoptosis in siRNA-transfected keratinocytes. (A) Sub-confluent keratinocytes were transfected with ZFP36- and ZFP36L1specific siRNAs, grown to confluence, and serum-starved for 48 h (Scale bar: 100 m). (B) Subsequently, cell proliferation was analyzed via Ki67 staining and analysis of Ki67 mRNA levels. In parallel, cells were stained with rhodamine-conjugated phalloidin, to study structural organization of the cytoskeleton. Representative pictures are shown. (C) After 48 h, expression of the genes encoding the cell cycle regulators cyclin A2 (CycA2) and cyclin-dependent kinase 1 (CDK1) was analyzed by qPCR. (D and E) Furthermore, cells in G2/M phase and the number of apoptotic cells was quantified via FACS sorting as indicated. (F) Finally, expression of the epidermal differentiation markers K10 and involucrin were analyzed in the transfected cells via immunofluorescence, qPCR and Western blot. Data are expressed as means +/− SD (n = 2: B, C and F; n = 3: D and E). *: p < 0.05; **: p < 0.01, ***: p < 0.001.
Please cite this article in press as: Prenzler, F., et al., Functional analysis of ZFP36 proteins in keratinocytes. Eur. J. Cell Biol. (2016), http://dx.doi.org/10.1016/j.ejcb.2016.04.007
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Fig. 5. Regulation of VEGF production in ZFP36 siRNA-transfected cells after scratching. After scratching, VEGF concentrations in conditioned medium were determined by ELISA analysis as indicated. The top graph represents the time course of VEGF levels in the conditioned medium of untransfected cells, incubated in fresh medium overnight (pre), and at the indicated time points after scratching. The bottom graphs show VEGF concentrations in the conditioned medium of siRNA-transfected cells, before scratching (left), and at defined time points after scratching (right). Data are expressed as mean +/− SD (n = 3). **: p < 0.01; ***: p < 0.001.
100 l extraction buffer (10 mM HEPES, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA pH 7.9) and incubated on ice for 10 min. Samples were then sonicated on ice for five 5-s-intervals, and after centrifugation, supernatants were collected. Protein concentration was determined using the BCA kit (Pierce), and samples were transferred onto nitrocellulose membranes and blocked and incubated with antibodies as described (Adams et al., 2007) using a monoclonal mouse antibody specific for ZFP36 (#ab119779; Abcam, Cambridge, UK), and polyclonal rabbit antibodies specific for ZFP36L1 (#ab42473; Abcam, Cambridge, UK), and ZFP36L2 (#ab70775; Abcam, Cambridge, UK). Incubation with a mouse anti-GAPDH antibody (R&D Systems, Minneapolis, USA), or a rabbit anti-GAPD antibody (Merck-Millipore, Schwalbach, Germany) served as a loading control. For detection, horseradish peroxidasecoupled anti-rabbit or anti-mouse secondary antibodies (Dianova, Hamburg, Germany) and the Amersham ECL detection system (GE Healthcare, Freiburg, Germany), or Li-Cor donkey anti-rabbit IgG 800 and Li-Cor anti-mouse IgG 680 antibodies in combination with a Li-Cor fluorescence imaging system (Li-Cor, Nebraska, USA), were employed.
body (clone SP6, Dianova, #KI68IC002) was used. F-actin was stained using rhodamine-labelled phalloidin. 2.8. FACS analysis For FACS analysis, keratinocytes were trypsinized, washed with PBS and fixed with 70% ethanol. Propidium iodide (PI) staining was carried out by incubating the cells in 1% PI in the dark. Subsequently, cells were FACS-sorted using a BD FACSCanto II flow cytometer (BD Biosciences, San Jose, CA, USA). The proportion of apoptotic cells was determined by quantification of hypodiploid nuclei, according to the method described by Riccardi and Nicoletti (2006). 2.9. VEGF ELISA VEGF concentrations in HaCaT cell supernatants were determined using the ELISA Duo Set in combination with an anti-VEGF antibody (R&D Systems, Minneapolis, USA). 3. Results 3.1. Localization of ZFP36, ZFP36L1, and ZFP36L2 in the skin
2.7. Immunofluorescence Immunofluorescence was carried out as previously described (Adams et al., 2007). Paraffin sections were deparaffinized, pretreated with citrate buffer, and stained using the protocol “NovoLink—Polymer Detection System” (Leica). For detection of ZFP36, ZFP36L1, and ZFP36L2, Abcam antibodies were used (see above). For keratin 14 (K14) and keratin 10 (K10) immunofluorescence staining, mouse monoclonal antibodies (Abcam #ab7800, and Progen, clone DE-K10, #11414, respectively) were employed. Nuclei were stained with DAPI (4 ,6-diamidin-2-phenylindol). For the detection of Ki67 in cultured cells, a mouse monoclonal anti-
In our previous study (Hacker et al., 2010), we could demonstrate induction of all three ZFP36 genes after scratch-wounding of human HaCaT keratinocytes in vitro. In addition, after wounding of murine skin in vivo, we could detect upregulation of ZFP36 expression. However, still, the spatial expression patterns of the three ZFP36 genes in the skin remained unclear, specifically, it was not known whether the genes were expressed in the dermal or the epidermal compartment or both. Thus, we aimed at elucidating the spatial expression patterns of the three ZFP36 genes in the skin. For this purpose, immunohistochemical studies were carried out. Since we could not obtain
Please cite this article in press as: Prenzler, F., et al., Functional analysis of ZFP36 proteins in keratinocytes. Eur. J. Cell Biol. (2016), http://dx.doi.org/10.1016/j.ejcb.2016.04.007
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specific immunohistochemical staining for all ZFP36 proteins on murine skin sections, we focused on human skin. As shown in Fig. 1, in normal belly skin, ZFP36 was found in single cells of both the epidermal basal and suprabasal layers, whereas ZFP36L1 and ZFP36L2 were present in almost all cells of the basal and – at lower levels – suprabasal layers. These findings were also confirmed by co-staining for keratin K14 and K10, which are present in the basal or suprabasal epidermal layers, respectively (Fuchs, 1991; Fig. 1 and data not shown). Furthermore, our data indicate that in contrast to ZFP36L1 and L2, ZFP36 is predominantly nuclear in epidermal keratinocytes. In addition, at significantly lower levels, all three ZFP36 proteins could also be detected in the dermal compartment. This finding corresponds to qPCR results obtained when analyzing the dermal-epidermal expression of the three ZFP36 genes in murine skin (data not shown), indicating that fundamental differences between human and murine skin with respect to the dermal-epidermal distribution of the three ZFP36 proteins are unlikely. Taken together, these data suggest that all three ZFP36 genes are expressed in both the dermal and the epidermal compartments in human as well as in murine skin.
3.2. Expression of ZFP36, ZFP36L1, and ZFP36L2 in scratched keratinocytes at the protein level Since our previous study (Hacker et al., 2010) had only included RNA data on the expression of the ZFP36 genes after scratchwounding, we analyzed ZFP36, ZFP36L1 and ZFP36L2 protein levels in quiescent, scratch-wounded HaCaT keratinocytes. As shown in Fig. 2, as suggested by the RNA data, we could detect induction of ZFP36 gene expression after scratching also at the protein level. By contrast, presumably due to insufficient sensitivity of the Western blot analysis and in contrast to the RNA data, we could not detect an increase in ZFP36L1 or ZFP36L2 protein levels after scratching. For these two isoforms, induction of the respective genes had already been less pronounced at the mRNA level (Hacker et al., 2010; Fig. 2).
3.3. Inhibition of ZFP36 and ZFP36L1 gene expression in cultured keratinocytes For a further functional analysis, we focused on ZFP36 and ZFP36L1. For this purpose, cultured HaCaT cells were transfected with specific siRNAs. In the case of ZFP36L1, this technology has already successfully been employed in our previous study (Hacker et al., 2010), and the efficiency of siRNA transfection was also confirmed via FACS analysis of cells that had been transfected with a FITC-labelled, unspecific (“scrambled”) siRNA control (Fig. 3A). Using this approach, at the mRNA level, we could achieve an 80–90% reduction in ZFP36 and ZFP36L1 transcript levels when compared to cells that had been transfected with an unspecific (“scrambled”, scr) siRNA control (Fig. 3B). To rule out a potential cross-regulation between the two isoforms, which might hamper subsequent functional studies, expression of the respective other two ZFP36 gene family members was analyzed in the siRNA-transfected cells, too. As shown in Fig. 3B, transfection with a specific siRNA directed against ZFP36 or ZFP36L1 had no or only minor effects on the expression of the genes encoding the other variant, neither directly after transfection nor after serum starvation or during a subsequent scratch-wounding protocol. These results indicate that, as already suggested by our previous data obtained with a ZFP36L1 siRNA transfection model system (Hacker et al., 2010), there is no significant cross-regulation between the genes encoding ZFP36 and ZFP36L1.
3.4. Analysis of proliferation, differentiation, and apoptosis in siRNA-transfected keratinocytes Remarkably, when identical numbers of transfected cells and controls were seeded, cells in which ZFP36 or ZFP36L1 expression had been inhibited appeared less dense in contrast to scr-transfected controls after serum withdrawal. In addition, morphologically, cells showed a more heterogeneous appearance: Some were spindle-shaped, some were more rounded, reminiscent of an apoptotic phenotype (Fig. 4A). Thus, we addressed the question whether blockade of ZFP36 and ZFP36L1 gene expression influences keratinocyte proliferation, differentiation, or apoptosis. Firstly, siRNA-transfected cells were stained with an antibody directed against Ki-67, a nuclear marker for proliferating cells. The cells’ cytoplasm was counterstained with rhodamine-conjugated phalloidin, directed against the cytoskeletal F-actin protein. As shown in Fig. 4B, we could demonstrate a decreased percentage of Ki-67-positive nuclei in the cells that had been transfected with the ZFP36- or the ZFP36L1-specific siRNAs, whereas the intracellular F-actin distribution was not altered, indicating no changes with respect to cytoskeletal organization. In addition, Ki67 expression was strongly decreased at the mRNA level (Fig. 4B), whereas at least in the case of the ZFP36L1 siRNA-transfected cells, expression of the gene encoding the cell cycle inhibitor p21 was moderately enhanced (data not shown). To test the hypothesis that ZFP36 and/or ZFP36L1 might be involved in cell cycle control, we studied expression of the genes encoding the cell cycle regulators cyclin A2 (CycA2) and cyclin-dependent kinase 1 (CDK1) in the siRNAtransfected cells and found that both genes were downregulated in the ZFP36- and particularly in the ZFP36L1 siRNA-transfected cells, indicating a role for both ZFP36 proteins in the regulation of keratinocyte cell cycle and proliferation (Fig. 4C). Furthermore, we analyzed the possibility that the siRNA-transfected cells might show abnormalities with respect to cell cycle progression using FACS analysis. Indeed, as shown in Fig. 4D, for the ZFP36L1 siRNAtransfected keratinocytes, we could demonstrate an increased percentage of cells in G2/M phase when compared to scr siRNAtransfected controls. This phenomenon could not be observed when analyzing the cells that had been transfected with the ZFP36 siRNA. In addition, a potential effect on apoptosis in the cells that had been transfected with the ZFP36- and ZFP36L1-specific siRNAs was further studied via FACS analysis. As shown in Fig. 4E, using this approach, we could demonstrate enhanced apoptosis in the cells that had been transfected with the ZFP36L1 siRNA both in serumcontaining and in serum-free medium. At later time points and in serum-free medium, a slightly enhanced percentage of apoptotic cells could also be observed after transfection with the ZFP36 siRNA (Fig. 4E). Moreover, we analyzed the expression of the keratinocyte differentiation marker keratin 10 (K10) in our siRNA-transfected cells. As shown in Fig. 4F, we found a higher percentage of cells positive for the suprabasal marker K10 in the batches that had been transfected with the ZFP36-specific siRNA, indicating that this factor might also be involved in the regulation of keratinocyte differentiation. However, it should be mentioned that HaCaT cells do not possess full keratinocyte differentiation potential (Ryle et al., 1989), and that furthermore, K10 expression in cultured keratinocytes is regulated in a complex and multifactorial manner, at both the mRNA and the protein level, thus, these data might not be representative for the in vivo situation, where K10 levels are highest in early-differentiating cells (Borowiec et al., 2013). In addition, we did not find elevated concentrations of involucrin, an “early” keratinocyte differentiation marker (Watt, 1983), after ZFP36 or ZFP36L1 siRNA transfection (Fig. 4F and data not shown).
Please cite this article in press as: Prenzler, F., et al., Functional analysis of ZFP36 proteins in keratinocytes. Eur. J. Cell Biol. (2016), http://dx.doi.org/10.1016/j.ejcb.2016.04.007
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3.5. Scratch-wounding and reepithelialization of siRNA-transfected keratinocyte monolayers Given the effects of ZFP36 and ZFP36L1 depletion on keratinocyte proliferation, differentiation and apoptosis, it appeared likely that these factors might also influence the velocity of wound closure. Thus, to study the effects of ZFP36 and ZFP36L1 inhibition on epidermal wound healing, siRNA-transfected keratinocyte monolayers were scratch-wounded as previously described (Hacker et al., 2010). The reepithelialization process was documented photographically and subsequently analyzed and quantified (Turchi et al., 2002). However, the velocity of reepithelialization in ZFP36and ZFP36L1-siRNA-transfected cells was not significantly altered when compared to cells that had been transfected with an unspecific (scr) control (data not shown). 3.6. Regulation of growth factor and cytokine production in siRNA-transfected cells after wounding Since epidermal morphogenesis and cutaneous wound repair are particularly regulated by growth factors and cytokines, and since ZFP36 proteins, via destabilization of the respective mRNAs, specifically influence the production of these factors, it is likely that ZFP36 proteins are involved in the modulation of keratinocyte growth factor and cytokine production. Since we had previously shown that depletion of ZFP36L1 from HaCaT cells enhanced keratinocyte VEGF, but not TNF-␣ production (Hacker et al., 2010), we tested the hypothesis that the VEGF transcript might also be a target of ZFP36 in these cells. For this purpose, we determined VEGF concentrations in conditioned medium of cells that had been transfected with ZFP36 siRNA, brought to quiescence, and subsequently scratch-wounded. As shown in Fig. 5, using this approach, we found that steady-state VEGF concentrations were already elevated in ZFP36 siRNA-transfected cells before scratching when compared to cells that had been transfected with an unspecific (scr) control. After a change of medium and scratching, the increase in extracellular VEGF concentrations was much faster in the ZFP36 siRNA-transfected cells when compared to controls, indicating that keratinocyte-derived VEGF is indeed regulated by both ZFP36 and ZFP36L1. 4. Discussion When studying the localization of ZFP36, ZFP36L1, and ZFP36L2 in human and murine skin, we found that all three proteins were present in both the epidermal the dermal compartments. Nevertheless, intra-epidermally, the localization of ZFP36 differed from that of the other two isoforms: Whereas ZFP36L1 and ZFP36L2 could be detected in almost all cells and particularly in the basal, proliferating layer, ZFP36 could be found in approximately 50% of the cells all over the epidermis, both in basal and in suprabasal keratinocytes. In addition, our immunofluorescence studies indicate that epidermal ZFP36 might be predominantly nuclear. This is consistent with earlier reports for other cell types, demonstrating that in unstimulated cells, ZFP36 is mostly nuclear, and translocates to the cytoplasm upon growth factor or cytokine stimulation (for review, see Baou et al., 2009). After scratch-wounding, we found that, consistent with our previous data (Hacker et al., 2010), all ZFP36 genes were induced at the mRNA level, however, at the protein level, induction was only detectable for the ZFP36 gene. One explanation for this might be the fact that induction of the other two genes was not as pronounced at the mRNA level, thus not allowing the detection of subtle changes at the protein level. In addition, despite the fact that we could previously demonstrate quite similar expression kinetics for the three
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ZFP36 genes in HaCaT cells and in primary keratinocytes (Hacker et al., 2010), it is possible that we might have obtained different results in the latter. Finally, the un-physiological, non-stratified nature of our cultures might have influenced our results. Thus, in the future, experiments in a “stratified” in vitro model system, might be interesting. After inhibition of ZFP36 and ZFP36L1 expression in keratinocytes using specific siRNAs, consistent with previous reports analyzing other cell types and tissues, no cross-regulation could be detected between the two genes. These data suggest that there might be little functional redundancy between ZFP36 and ZFP36L1 in keratinocytes. The fact that all three ZFP36 proteins might each have physiologically unique functions in general has already been suggested by the impressively unique, non-overlapping phenotypes of the respective knockout animals (for review, see Baou et al., 2009; Sanduja et al., 2011; Ciais et al., 2013) After inhibition of both ZFP36 and ZFP36L1 expression, the number of Ki67-positive cells was reduced, indicating that overall, the proportion of cells within an active phase of the cell cycle was decreased. However, upon inhibition of at least ZFP36L1 expression, we found an increased proportion of cells in the G2/M phase of the cell cycle, indicating G2/M phase arrest. It is likely that this is at least in part due to effects on mRNAs encoding cell cycle regulators, such as p21, or PLK3 (polo-like kinase 3), which have been shown to be targets of ZFP36 (for review, see Baou et al., 2009; Sanduja et al., 2011) and might also represent targets of ZFP36L1. In this context, it is interesting that p21 has been shown to potentiate G2/M arrest (for review, see Stark and Taylor, 2006), and it is likely that PLK3 also functions as enhancer of this process (for review, see Strebhardt, 2010). Against this background, it is unclear why we could not detect effects of ZFP36 depletion on G2/M arrest. Potential explanations might be that inhibition of ZFP36 expression was slightly less pronounced than that of ZFP36L1 expression, or that effects of ZFP36 on other targets or non mRNA target-associated effects of ZFP36 might overpower potential effects on G2/M arrest. Consistent with the fact that cells arrested in G2/M are committed to the apoptotic pathway (for review, see Rieder, 2011), we also found a higher proportion of apoptotic cells after inhibition of ZFP36L1 expression. Interestingly, we also observed an increased percentage of cells positive for the differentiation-specific K10 after ZFP36 siRNA transfection, suggesting that ZFP36 might inhibit the expression of some keratinocyte differentiation markers. Despite the fact that we found profound effects of ZFP36 and ZFP36L1 siRNA-mediated downregulation on keratinocyte proliferation, apoptosis, and differentiation, re-epithelialization of scratch wounds was not affected. However, we observed an overall lower density of the transfected cells when compared to controls, specifically in serum-free medium, even before scratching, irrespective of the fact that initially, identical cell numbers had been seeded. Thus, since overall re-epithelialization of the scratch wounds was quantified via determining the area of re-coverage (or, rather, the remaining cell-free area of the scratch), it is likely that the low amount of untransfected cells still present in our cultures was sufficient to bring about re-epithelialization at a similar speed as in controls, albeit overall cell density was lower. Nevertheless, most importantly, to better understand a potential role of ZFP36 proteins in wound healing, the results will have to be reproduced in an in vivo model system, e.g. epidermis-specific knockout animals. Since we could previously demonstrate that ZFP36L1 was a potent regulator of VEGF transcript levels in keratinocytes (Hacker et al., 2010), we also analyzed VEGF production in HaCaT cells after inhibition of ZFP36 expression. As expected, we found that decreased ZFP36 levels also enhanced VEGF production in keratinocytes, indicating that both ZFP36 and ZFP36L1 might con-
Please cite this article in press as: Prenzler, F., et al., Functional analysis of ZFP36 proteins in keratinocytes. Eur. J. Cell Biol. (2016), http://dx.doi.org/10.1016/j.ejcb.2016.04.007
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tribute to the increase in epidermal VEGF release after wounding (Brown et al., 1992), and thus promote angiogenesis during healing. Acknowledgements We thank Sabine Werner, ETH Zurich, for RNA and tissue sections. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (Mu 1556/5-1) (to B.M.). References Adams, S., Pankow, S., Werner, S., Munz, B., 2007. Regulation of NF-B activity and keratinocyte differentiation by the RIP4 protein: implications for cutaneous wound repair. J. Invest. Dermatol. 127, 538–544. Bakheet, T., Frevel, M., Williams, B.R., Greer, W., Khabar, K.S., 2001. ARED: human AU-rich element-containing mRNA database reveals an unexpectedly diverse functional repertoire of encoded proteins. Nucleic Acids Res. 29, 246–254. Baou, M., Jewell, A., Murphy, J.J., 2009. TIS11 family proteins and their roles in posttranscriptional gene regulation. J. Biomed. Biotechnol., http://dx.doi.org/ 10.1155/2009/634520. Bell, S.E., Sanchez, M.J., Spasic-Boskovic, O., Santalucia, T., Gambardella, L., Burton, G.J., Murphy, J.J., Norton, J.D., Clark, A.R., Turner, M., 2006. The RNA binding protein Zfp36l1 is required for normal vascularisation and post-transcriptionally regulates VEGF expression. Dev. Dyn. 235, 3144–3155. Blackshear, P.J., Phillips, R.S., Ghosh, S., Ramos, S.B., Richfield, E.K., Lai, W.S., 2005. Zfp36l3, a rodent X chromosome gene encoding a placenta-specific member of the Tristetraprolin family of CCCH tandem zinc finger proteins. Biol. Reprod. 73, 1072. Borowiec, A.S., Delcourt, P., Dewailly, E., Bidaux, G., 2013. Optimal differentiation of in vitro keratinocytes requires multifactorial external control. PLoS One 8 (October (10)), e77507, http://dx.doi.org/10.1371/journal.pone.0077507. eCollection 2013. Brown, L.F., Yeo, K.T., Berse, B., Yeo, T.K., Senger, D.R., Dvorak, H.F., van de Water, L., 1992. Expression of vascular permeability factor (vascular endothelial growth factor) by epidermal keratinocytes during wound healing. J. Exp. Med. 176, 1375–1379. Busse, M., Schwarzburger, M., Berger, F., Hacker, C., Munz, B., 2008. TIS11B is a regulator of myogenic differentiation. Eur. J. Cell Biol. 87, 163–172. Caput, D., Beutler, B., Hartog, K., Thayer, R., Brown-Shimer, S., Cerami, A., 1986. Identification of a common nucleotide sequence in the 3 -untranslated region of mRNA molecules specifying inflammatory mediators. Proc. Natl. Acad. Sci. U. S. A. 83, 1670–1674. Carballo, E., Lai, W.S., Blackshear, P.J., 2000. Evidence that tristetraprolin is a physiological regulator of granulocyte-macrophage colony-stimulating factor messenger RNA deadenylation and stability. Blood 95, 1891–1899. Carrick, D.M., Blackshear, P.J., 2007. Comparative expression of tristetraprolin (TTP) family member transcripts in normal human tissues and cancer cell lines. Arch. Biochem. Biophys. 462, 278–285. Ciais, D., Cherradi, N., Bailly, S., Grenier, E., Berra, E., Pouyssegur, J., Lamarre, J., Feige, J.J., 2004. Destabilization of vascular endothelial growth factor mRNA by the zinc-finger protein TIS11b. Oncogene 23, 8673–8680. Ciais, D., Cherradi, N., Feige, J.-J., 2013. Multiple functions of tristetraprolin/TTP RNA-binding proteins in the regulation of mRNA biogenesis and degradation. Cell. Mol. Life Sci. 70, 2031–2044. DuBois, R.N., Bishop, P.R., Graves-Deal, R., Coffey, R.J., 1995. Transforming growth factor alpha regulation of two zinc finger-containing immediate early response genes in intestine. Cell Growth Differ. 6, 523–529. Emmons, J., Townley-Tilson, W.H., Deleault, K.M., Skinner, S.J., Gross, R.H., Whitfield, M.L., Brooks, S.A., 2008. Identification of TTP mRNA targets in human dendritic cells reveals TTP as a critical regulator of dendritic cell maturation. RNA 14, 888–902. Fuchs, E., 1991. Keratin genes, epidermal differentiation and animal models for the study of human skin diseases. Biochem. Soc. Trans. 19, 1112–1115. Gomperts, M., Corps, A.N., Pascall, J.C., Brown, K.D., 1992. Mitogen-induced expression of the primary response gene cMG1 in a rat intestinal epithelial cell line (RIE-1). FEBS Lett. 306, 1–4. Gurtner, G.C., Werner, S., Barrandon, Y., Longaker, M.T., 2008. Wound repair and regeneration. Nature 453, 314–321. Hacker, C., Valchanova, R., Adams, S., Munz, B., 2010. ZFP36L1 is regulated by growth factors and cytokines in keratinocytes and influences their VEGF production. Growth Factors 28, 178–190.
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Research paper
Functional analysis of ZFP36 proteins in keratinocytes Frauke Prenzler, Annunziata Fragasso, Angelika Schmitt, Barbara Munz ∗ University Hospital Tübingen, Medical Clinic, Department of Sports Medicine, Hoppe-Seyler-Str. 6, D-72076 Tübingen, Germany
a r t i c l e
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Article history: Received 13 July 2015 Received in revised form 28 April 2016 Accepted 28 April 2016 Keywords: ZFP36 proteins Epidermis Keratinocytes
a b s t r a c t The ZFP36 family of zinc finger proteins, including ZFP36, ZFP36L1, and ZFP36L2, regulates the production of growth factors and cytokines via destabilization of the respective mRNAs. We could recently demonstrate that in cultured keratinocytes, expression of the ZFP36, ZFP36L1, and ZFP36L2 genes is induced by growth factors and cytokines and that ZFP36L1 is a potent regulator of keratinocyte VEGF production. We now further analyzed the localization and function of ZFP36 proteins in the skin, specifically in epidermal keratinocytes. We found that in human epidermis, the ZFP36 protein could be detected in basal and suprabasal keratinocytes, whereas ZFP36L1 and ZFP36L2 were expressed mainly in the basal layer, indicating different and non-redundant functions of the three proteins in the epidermis. Consistently, upon inhibition of ZFP36 or ZFP36L1 expression using specific siRNAs, there was no major effect on expression of the respective other gene. In addition, we demonstrate that both ZFP36 and ZFP36L1 influence keratinocyte cell cycle, differentiation, and apoptosis in a distinct manner. Finally, we show that similarly as ZFP36L1, ZFP36 is a potent regulator of keratinocyte VEGF production. Thus, it is likely that both proteins regulate angiogenesis via paracrine mechanisms. Taken together, our results suggest that ZFP36 proteins might control reepithelialization and angiogenesis in the skin in a multimodal manner. © 2016 Elsevier GmbH. All rights reserved.
1. Introduction Cutaneous wound repair is a highly complex process involving many different cell types, such as keratinocytes, fibroblasts, endothelial cells, and immune cells. The different stages of healing—inflammation, reepithelialization, contraction and angiogenesis—are regulated by a multitude of growth factors and cytokines, such as EGF, TGF, FGF1, KGF, HGF, VEGF, and TNF␣. The epidermis, which forms the outer layer of the skin, consists mainly of keratinocytes. They differentiate from the basal layer, however, during wound healing, epidermal keratinocytes migrate into the wound site and become hyperproliferative (reviewed by Martin, 1997; Gurtner et al., 2008). ZFP36 (tristetraprolin TTP, Tis11, Nup34), ZFP36L1 (Tis11B, ERF1, BRF1, berg36), and ZFP36L2 (Tis11D, ERF2, BRF2) are members of the ZFP36 family of RNA-binding zinc finger proteins (DuBois et al., 1995; Varnum et al., 1991). They appear to be evolutionary “old” proteins, since ZFP36 proteins have been identified in a broad variety of species from yeast (Ma and Herschman, 1995) to
∗ Corresponding author. E-mail address: [email protected] (B. Munz).
humans (Taylor et al., 1991), except for the ZFP36L3 isoform, which seems to be mouse-specific (Blackshear et al., 2005). ZFP36 proteins bind to and destabilize specific target mRNAs, such as the TNF˛, the GM-CSF, and the VEGF transcripts, thus reducing expression of the respective genes. Recognition occurs via binding of the ZFP36 proteins to AU-rich elements (AREs) within the target transcripts’ 3 untranslated regions (3 UTRs) (Lai et al., 2000; Carballo et al., 2000; Ciais et al., 2004; for review, see Baou et al., 2009; Sanduja et al., 2011; Ciais et al., 2013). Despite the fact that AREs were first discovered in cytokine and protooncogene sequences (Caput et al., 1986), genome-wide screens have identified AREs in a broad variety of different mRNAs encoding proteins of multiple and diverse functions (Bakheet et al., 2001; Lai et al., 2006). Expression of the ZFP36/Zfp36 (human/rodent), ZFP36L1/Zfp36l1 and ZFP36L2/Zfp36l2 genes can be induced by various stimuli, e.g. growth factors, in a broad range of cell types (Varnum et al., 1989a,b; Gomperts et al., 1992; Reppe et al., 2004; Manabe et al., 1999). All three genes are expressed in a huge variety of cell types and tissues, however, each has its unique and distinct spatial and temporal expression pattern (Carrick and Blackshear, 2007). With respect to the physiological role of the ZFP36 proteins under in vivo conditions, studies in knock-out mice demonstrated roles
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Fig. 1. Localization of ZFP36, ZFP36L1, and ZFP36L2 in the skin. Expression of the three ZFP36 genes was analyzed in sections from human belly skin as indicated. Co-staining was performed with an antibody directed against the proliferation -associated keratin 14. Nuclei were stained with DAPI.
Fig. 2. Expression of ZFP36 and ZFP36L1 after scratch-wounding at the RNA and at the protein level. qPCR and Western blot analysis were performed on RNA and protein samples derived from quiescent, scratch-wounded keratinocyte monolayers as indicated. The blots were also probed with an antibody directed against GAPDH as a loading control and a reference for normalization. Data are expressed as means +/− SD (n = 5). *: p < 0.05; **: p < 0.01.
for ZFP36, ZFP36L1, and ZFP36L2, in inflammation, chorioallantoic fusion and vascularization, and early embryonic development, respectively (Taylor et al., 1996; Stumpo et al., 2004; Ramos et al., 2004; Bell et al., 2006). In addition, ZFP36L1 has been implicated in myogenesis (Busse et al., 2008), and ZFP36 has been identified as regulator of dendritic cell maturation (Emmons et al., 2008), but little is known so far about a possible role of ZFP36 proteins in keratinocytes or in cutaneous wound repair.
We could recently demonstrate induction of all three ZFP36 genes, specifically of ZFP36 and ZFP36L1, after scratch-wounding in vitro. In addition, we could show that ZFP36L1 regulates keratinocyte VEGF production, thereby potentially modulating the angiogenic response in wound tissue (Hacker et al., 2010). Furthermore, most interestingly, a recent report demonstrated that the anti-inflammatory effect of glucocorticoids on the epidermis is in part mediated by ZFP36 (Sevilla et al., 2015).
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Fig. 3. Inhibition of ZFP36, ZFP36L1, and ZFP36L2 gene expression in cultured keratinocytes. HaCaT keratinocytes were transfected with specific siRNAs directed against ZFP36 or ZFP36L1, an unspecific (“scrambled”) negative control, or left untreated. (A) To assess the efficiency of the transfection procedure, the cells were first transfected with a FITC-labelled “scrambled” siRNA and analyzed by FACS sorting. (B) After inhibition of expression of ZFP36 or ZFP36L1 via siRNA transfection, cells were rendered quiescent by serum starvation and subsequently scratch-wounded. Expression of ZFP36 and ZFP36L1 was analyzed by qPCR. Data are expressed as means +/− SD (n = 2). *: p < 0.05; **: p < 0.01. (C) ZFP36L1 expression was also analyzed by Western blot 24 h after transfection (without scratching). GAPDH levels were assessed as a control for equal loading.
Now, we functionally analyzed the role of the three ZFP36 proteins in epidermal keratinocytes. Specifically, we demonstrate that each of the three genes has its specific and unique spatial expression pattern in the epidermis and that inhibiting ZFP36 or ZFP36L1 expression in cultured keratinocytes induces apoptosis, blocks cell proliferation, and enhances VEGF production. Our data indicate that ZFP36 proteins might be multi-functional players in the skin and perhaps also in wound healing.
2. Experimental procedures 2.1. Skin tissue Paraffin sections from human belly skin were purchased from Zyagen, San Diego, CA, USA.
2.2. Cell culture Human HaCaT keratinocytes were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum.
2.4. Transfection of keratinocytes with siRNA 1 × 106 HaCaT keratinocytes per well were trypsinized, washed, resuspended and reverse-transfected with ZFP36-, or ZFP36L1specific siRNA or an unspecific (“scrambled”, scr) siRNA as a negative control (final concentration of all siRNAs: 100 nM). 8 l Lipofectamine (Life Technologies) were used as a transfection reagent. After incubation in 6-well plates at 37 ◦ C for 24 h, the medium was replaced by regular or serum-free medium. 2.5. RNA isolation and qPCR RNA isolation was performed using the AllPrep kit (Qiagen). Semi-quantitative real time PCR analysis was carried out using the iCycler MyiQ system (Bio-Rad). Gene expression was analyzed using the Eva Green Mastermix (Bio-Rad). For detection of different transcripts, pre-designed primers (Qiagen QuantiTect Primer Assays) were used. In each experiment, melting curve analysis was performed to verify that a single transcript was produced. RT-qPCR relative gene expression was calculated using the comparative CT (2−DC T ) method, where expression was normalized to RPL13 and GAPDH. Non-RT- and non-template controls were run for all reactions.
2.3. Scratch-wounding 2.6. Generation of protein extracts and Western blotting HaCaT cells were grown in 6-well plates and serum-starved overnight. They were then scratched with a scalpel to yield wounds with uniform width.
Whole cell protein lysates were generated as follows: cells were harvested in PBS and cell pellets were resuspended in
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Fig. 4. Analysis of proliferation, differentiation, and apoptosis in siRNA-transfected keratinocytes. (A) Sub-confluent keratinocytes were transfected with ZFP36- and ZFP36L1specific siRNAs, grown to confluence, and serum-starved for 48 h (Scale bar: 100 m). (B) Subsequently, cell proliferation was analyzed via Ki67 staining and analysis of Ki67 mRNA levels. In parallel, cells were stained with rhodamine-conjugated phalloidin, to study structural organization of the cytoskeleton. Representative pictures are shown. (C) After 48 h, expression of the genes encoding the cell cycle regulators cyclin A2 (CycA2) and cyclin-dependent kinase 1 (CDK1) was analyzed by qPCR. (D and E) Furthermore, cells in G2/M phase and the number of apoptotic cells was quantified via FACS sorting as indicated. (F) Finally, expression of the epidermal differentiation markers K10 and involucrin were analyzed in the transfected cells via immunofluorescence, qPCR and Western blot. Data are expressed as means +/− SD (n = 2: B, C and F; n = 3: D and E). *: p < 0.05; **: p < 0.01, ***: p < 0.001.
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Fig. 5. Regulation of VEGF production in ZFP36 siRNA-transfected cells after scratching. After scratching, VEGF concentrations in conditioned medium were determined by ELISA analysis as indicated. The top graph represents the time course of VEGF levels in the conditioned medium of untransfected cells, incubated in fresh medium overnight (pre), and at the indicated time points after scratching. The bottom graphs show VEGF concentrations in the conditioned medium of siRNA-transfected cells, before scratching (left), and at defined time points after scratching (right). Data are expressed as mean +/− SD (n = 3). **: p < 0.01; ***: p < 0.001.
100 l extraction buffer (10 mM HEPES, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA pH 7.9) and incubated on ice for 10 min. Samples were then sonicated on ice for five 5-s-intervals, and after centrifugation, supernatants were collected. Protein concentration was determined using the BCA kit (Pierce), and samples were transferred onto nitrocellulose membranes and blocked and incubated with antibodies as described (Adams et al., 2007) using a monoclonal mouse antibody specific for ZFP36 (#ab119779; Abcam, Cambridge, UK), and polyclonal rabbit antibodies specific for ZFP36L1 (#ab42473; Abcam, Cambridge, UK), and ZFP36L2 (#ab70775; Abcam, Cambridge, UK). Incubation with a mouse anti-GAPDH antibody (R&D Systems, Minneapolis, USA), or a rabbit anti-GAPD antibody (Merck-Millipore, Schwalbach, Germany) served as a loading control. For detection, horseradish peroxidasecoupled anti-rabbit or anti-mouse secondary antibodies (Dianova, Hamburg, Germany) and the Amersham ECL detection system (GE Healthcare, Freiburg, Germany), or Li-Cor donkey anti-rabbit IgG 800 and Li-Cor anti-mouse IgG 680 antibodies in combination with a Li-Cor fluorescence imaging system (Li-Cor, Nebraska, USA), were employed.
body (clone SP6, Dianova, #KI68IC002) was used. F-actin was stained using rhodamine-labelled phalloidin. 2.8. FACS analysis For FACS analysis, keratinocytes were trypsinized, washed with PBS and fixed with 70% ethanol. Propidium iodide (PI) staining was carried out by incubating the cells in 1% PI in the dark. Subsequently, cells were FACS-sorted using a BD FACSCanto II flow cytometer (BD Biosciences, San Jose, CA, USA). The proportion of apoptotic cells was determined by quantification of hypodiploid nuclei, according to the method described by Riccardi and Nicoletti (2006). 2.9. VEGF ELISA VEGF concentrations in HaCaT cell supernatants were determined using the ELISA Duo Set in combination with an anti-VEGF antibody (R&D Systems, Minneapolis, USA). 3. Results 3.1. Localization of ZFP36, ZFP36L1, and ZFP36L2 in the skin
2.7. Immunofluorescence Immunofluorescence was carried out as previously described (Adams et al., 2007). Paraffin sections were deparaffinized, pretreated with citrate buffer, and stained using the protocol “NovoLink—Polymer Detection System” (Leica). For detection of ZFP36, ZFP36L1, and ZFP36L2, Abcam antibodies were used (see above). For keratin 14 (K14) and keratin 10 (K10) immunofluorescence staining, mouse monoclonal antibodies (Abcam #ab7800, and Progen, clone DE-K10, #11414, respectively) were employed. Nuclei were stained with DAPI (4 ,6-diamidin-2-phenylindol). For the detection of Ki67 in cultured cells, a mouse monoclonal anti-
In our previous study (Hacker et al., 2010), we could demonstrate induction of all three ZFP36 genes after scratch-wounding of human HaCaT keratinocytes in vitro. In addition, after wounding of murine skin in vivo, we could detect upregulation of ZFP36 expression. However, still, the spatial expression patterns of the three ZFP36 genes in the skin remained unclear, specifically, it was not known whether the genes were expressed in the dermal or the epidermal compartment or both. Thus, we aimed at elucidating the spatial expression patterns of the three ZFP36 genes in the skin. For this purpose, immunohistochemical studies were carried out. Since we could not obtain
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specific immunohistochemical staining for all ZFP36 proteins on murine skin sections, we focused on human skin. As shown in Fig. 1, in normal belly skin, ZFP36 was found in single cells of both the epidermal basal and suprabasal layers, whereas ZFP36L1 and ZFP36L2 were present in almost all cells of the basal and – at lower levels – suprabasal layers. These findings were also confirmed by co-staining for keratin K14 and K10, which are present in the basal or suprabasal epidermal layers, respectively (Fuchs, 1991; Fig. 1 and data not shown). Furthermore, our data indicate that in contrast to ZFP36L1 and L2, ZFP36 is predominantly nuclear in epidermal keratinocytes. In addition, at significantly lower levels, all three ZFP36 proteins could also be detected in the dermal compartment. This finding corresponds to qPCR results obtained when analyzing the dermal-epidermal expression of the three ZFP36 genes in murine skin (data not shown), indicating that fundamental differences between human and murine skin with respect to the dermal-epidermal distribution of the three ZFP36 proteins are unlikely. Taken together, these data suggest that all three ZFP36 genes are expressed in both the dermal and the epidermal compartments in human as well as in murine skin.
3.2. Expression of ZFP36, ZFP36L1, and ZFP36L2 in scratched keratinocytes at the protein level Since our previous study (Hacker et al., 2010) had only included RNA data on the expression of the ZFP36 genes after scratchwounding, we analyzed ZFP36, ZFP36L1 and ZFP36L2 protein levels in quiescent, scratch-wounded HaCaT keratinocytes. As shown in Fig. 2, as suggested by the RNA data, we could detect induction of ZFP36 gene expression after scratching also at the protein level. By contrast, presumably due to insufficient sensitivity of the Western blot analysis and in contrast to the RNA data, we could not detect an increase in ZFP36L1 or ZFP36L2 protein levels after scratching. For these two isoforms, induction of the respective genes had already been less pronounced at the mRNA level (Hacker et al., 2010; Fig. 2).
3.3. Inhibition of ZFP36 and ZFP36L1 gene expression in cultured keratinocytes For a further functional analysis, we focused on ZFP36 and ZFP36L1. For this purpose, cultured HaCaT cells were transfected with specific siRNAs. In the case of ZFP36L1, this technology has already successfully been employed in our previous study (Hacker et al., 2010), and the efficiency of siRNA transfection was also confirmed via FACS analysis of cells that had been transfected with a FITC-labelled, unspecific (“scrambled”) siRNA control (Fig. 3A). Using this approach, at the mRNA level, we could achieve an 80–90% reduction in ZFP36 and ZFP36L1 transcript levels when compared to cells that had been transfected with an unspecific (“scrambled”, scr) siRNA control (Fig. 3B). To rule out a potential cross-regulation between the two isoforms, which might hamper subsequent functional studies, expression of the respective other two ZFP36 gene family members was analyzed in the siRNA-transfected cells, too. As shown in Fig. 3B, transfection with a specific siRNA directed against ZFP36 or ZFP36L1 had no or only minor effects on the expression of the genes encoding the other variant, neither directly after transfection nor after serum starvation or during a subsequent scratch-wounding protocol. These results indicate that, as already suggested by our previous data obtained with a ZFP36L1 siRNA transfection model system (Hacker et al., 2010), there is no significant cross-regulation between the genes encoding ZFP36 and ZFP36L1.
3.4. Analysis of proliferation, differentiation, and apoptosis in siRNA-transfected keratinocytes Remarkably, when identical numbers of transfected cells and controls were seeded, cells in which ZFP36 or ZFP36L1 expression had been inhibited appeared less dense in contrast to scr-transfected controls after serum withdrawal. In addition, morphologically, cells showed a more heterogeneous appearance: Some were spindle-shaped, some were more rounded, reminiscent of an apoptotic phenotype (Fig. 4A). Thus, we addressed the question whether blockade of ZFP36 and ZFP36L1 gene expression influences keratinocyte proliferation, differentiation, or apoptosis. Firstly, siRNA-transfected cells were stained with an antibody directed against Ki-67, a nuclear marker for proliferating cells. The cells’ cytoplasm was counterstained with rhodamine-conjugated phalloidin, directed against the cytoskeletal F-actin protein. As shown in Fig. 4B, we could demonstrate a decreased percentage of Ki-67-positive nuclei in the cells that had been transfected with the ZFP36- or the ZFP36L1-specific siRNAs, whereas the intracellular F-actin distribution was not altered, indicating no changes with respect to cytoskeletal organization. In addition, Ki67 expression was strongly decreased at the mRNA level (Fig. 4B), whereas at least in the case of the ZFP36L1 siRNA-transfected cells, expression of the gene encoding the cell cycle inhibitor p21 was moderately enhanced (data not shown). To test the hypothesis that ZFP36 and/or ZFP36L1 might be involved in cell cycle control, we studied expression of the genes encoding the cell cycle regulators cyclin A2 (CycA2) and cyclin-dependent kinase 1 (CDK1) in the siRNAtransfected cells and found that both genes were downregulated in the ZFP36- and particularly in the ZFP36L1 siRNA-transfected cells, indicating a role for both ZFP36 proteins in the regulation of keratinocyte cell cycle and proliferation (Fig. 4C). Furthermore, we analyzed the possibility that the siRNA-transfected cells might show abnormalities with respect to cell cycle progression using FACS analysis. Indeed, as shown in Fig. 4D, for the ZFP36L1 siRNAtransfected keratinocytes, we could demonstrate an increased percentage of cells in G2/M phase when compared to scr siRNAtransfected controls. This phenomenon could not be observed when analyzing the cells that had been transfected with the ZFP36 siRNA. In addition, a potential effect on apoptosis in the cells that had been transfected with the ZFP36- and ZFP36L1-specific siRNAs was further studied via FACS analysis. As shown in Fig. 4E, using this approach, we could demonstrate enhanced apoptosis in the cells that had been transfected with the ZFP36L1 siRNA both in serumcontaining and in serum-free medium. At later time points and in serum-free medium, a slightly enhanced percentage of apoptotic cells could also be observed after transfection with the ZFP36 siRNA (Fig. 4E). Moreover, we analyzed the expression of the keratinocyte differentiation marker keratin 10 (K10) in our siRNA-transfected cells. As shown in Fig. 4F, we found a higher percentage of cells positive for the suprabasal marker K10 in the batches that had been transfected with the ZFP36-specific siRNA, indicating that this factor might also be involved in the regulation of keratinocyte differentiation. However, it should be mentioned that HaCaT cells do not possess full keratinocyte differentiation potential (Ryle et al., 1989), and that furthermore, K10 expression in cultured keratinocytes is regulated in a complex and multifactorial manner, at both the mRNA and the protein level, thus, these data might not be representative for the in vivo situation, where K10 levels are highest in early-differentiating cells (Borowiec et al., 2013). In addition, we did not find elevated concentrations of involucrin, an “early” keratinocyte differentiation marker (Watt, 1983), after ZFP36 or ZFP36L1 siRNA transfection (Fig. 4F and data not shown).
Please cite this article in press as: Prenzler, F., et al., Functional analysis of ZFP36 proteins in keratinocytes. Eur. J. Cell Biol. (2016), http://dx.doi.org/10.1016/j.ejcb.2016.04.007
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3.5. Scratch-wounding and reepithelialization of siRNA-transfected keratinocyte monolayers Given the effects of ZFP36 and ZFP36L1 depletion on keratinocyte proliferation, differentiation and apoptosis, it appeared likely that these factors might also influence the velocity of wound closure. Thus, to study the effects of ZFP36 and ZFP36L1 inhibition on epidermal wound healing, siRNA-transfected keratinocyte monolayers were scratch-wounded as previously described (Hacker et al., 2010). The reepithelialization process was documented photographically and subsequently analyzed and quantified (Turchi et al., 2002). However, the velocity of reepithelialization in ZFP36and ZFP36L1-siRNA-transfected cells was not significantly altered when compared to cells that had been transfected with an unspecific (scr) control (data not shown). 3.6. Regulation of growth factor and cytokine production in siRNA-transfected cells after wounding Since epidermal morphogenesis and cutaneous wound repair are particularly regulated by growth factors and cytokines, and since ZFP36 proteins, via destabilization of the respective mRNAs, specifically influence the production of these factors, it is likely that ZFP36 proteins are involved in the modulation of keratinocyte growth factor and cytokine production. Since we had previously shown that depletion of ZFP36L1 from HaCaT cells enhanced keratinocyte VEGF, but not TNF-␣ production (Hacker et al., 2010), we tested the hypothesis that the VEGF transcript might also be a target of ZFP36 in these cells. For this purpose, we determined VEGF concentrations in conditioned medium of cells that had been transfected with ZFP36 siRNA, brought to quiescence, and subsequently scratch-wounded. As shown in Fig. 5, using this approach, we found that steady-state VEGF concentrations were already elevated in ZFP36 siRNA-transfected cells before scratching when compared to cells that had been transfected with an unspecific (scr) control. After a change of medium and scratching, the increase in extracellular VEGF concentrations was much faster in the ZFP36 siRNA-transfected cells when compared to controls, indicating that keratinocyte-derived VEGF is indeed regulated by both ZFP36 and ZFP36L1. 4. Discussion When studying the localization of ZFP36, ZFP36L1, and ZFP36L2 in human and murine skin, we found that all three proteins were present in both the epidermal the dermal compartments. Nevertheless, intra-epidermally, the localization of ZFP36 differed from that of the other two isoforms: Whereas ZFP36L1 and ZFP36L2 could be detected in almost all cells and particularly in the basal, proliferating layer, ZFP36 could be found in approximately 50% of the cells all over the epidermis, both in basal and in suprabasal keratinocytes. In addition, our immunofluorescence studies indicate that epidermal ZFP36 might be predominantly nuclear. This is consistent with earlier reports for other cell types, demonstrating that in unstimulated cells, ZFP36 is mostly nuclear, and translocates to the cytoplasm upon growth factor or cytokine stimulation (for review, see Baou et al., 2009). After scratch-wounding, we found that, consistent with our previous data (Hacker et al., 2010), all ZFP36 genes were induced at the mRNA level, however, at the protein level, induction was only detectable for the ZFP36 gene. One explanation for this might be the fact that induction of the other two genes was not as pronounced at the mRNA level, thus not allowing the detection of subtle changes at the protein level. In addition, despite the fact that we could previously demonstrate quite similar expression kinetics for the three
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ZFP36 genes in HaCaT cells and in primary keratinocytes (Hacker et al., 2010), it is possible that we might have obtained different results in the latter. Finally, the un-physiological, non-stratified nature of our cultures might have influenced our results. Thus, in the future, experiments in a “stratified” in vitro model system, might be interesting. After inhibition of ZFP36 and ZFP36L1 expression in keratinocytes using specific siRNAs, consistent with previous reports analyzing other cell types and tissues, no cross-regulation could be detected between the two genes. These data suggest that there might be little functional redundancy between ZFP36 and ZFP36L1 in keratinocytes. The fact that all three ZFP36 proteins might each have physiologically unique functions in general has already been suggested by the impressively unique, non-overlapping phenotypes of the respective knockout animals (for review, see Baou et al., 2009; Sanduja et al., 2011; Ciais et al., 2013) After inhibition of both ZFP36 and ZFP36L1 expression, the number of Ki67-positive cells was reduced, indicating that overall, the proportion of cells within an active phase of the cell cycle was decreased. However, upon inhibition of at least ZFP36L1 expression, we found an increased proportion of cells in the G2/M phase of the cell cycle, indicating G2/M phase arrest. It is likely that this is at least in part due to effects on mRNAs encoding cell cycle regulators, such as p21, or PLK3 (polo-like kinase 3), which have been shown to be targets of ZFP36 (for review, see Baou et al., 2009; Sanduja et al., 2011) and might also represent targets of ZFP36L1. In this context, it is interesting that p21 has been shown to potentiate G2/M arrest (for review, see Stark and Taylor, 2006), and it is likely that PLK3 also functions as enhancer of this process (for review, see Strebhardt, 2010). Against this background, it is unclear why we could not detect effects of ZFP36 depletion on G2/M arrest. Potential explanations might be that inhibition of ZFP36 expression was slightly less pronounced than that of ZFP36L1 expression, or that effects of ZFP36 on other targets or non mRNA target-associated effects of ZFP36 might overpower potential effects on G2/M arrest. Consistent with the fact that cells arrested in G2/M are committed to the apoptotic pathway (for review, see Rieder, 2011), we also found a higher proportion of apoptotic cells after inhibition of ZFP36L1 expression. Interestingly, we also observed an increased percentage of cells positive for the differentiation-specific K10 after ZFP36 siRNA transfection, suggesting that ZFP36 might inhibit the expression of some keratinocyte differentiation markers. Despite the fact that we found profound effects of ZFP36 and ZFP36L1 siRNA-mediated downregulation on keratinocyte proliferation, apoptosis, and differentiation, re-epithelialization of scratch wounds was not affected. However, we observed an overall lower density of the transfected cells when compared to controls, specifically in serum-free medium, even before scratching, irrespective of the fact that initially, identical cell numbers had been seeded. Thus, since overall re-epithelialization of the scratch wounds was quantified via determining the area of re-coverage (or, rather, the remaining cell-free area of the scratch), it is likely that the low amount of untransfected cells still present in our cultures was sufficient to bring about re-epithelialization at a similar speed as in controls, albeit overall cell density was lower. Nevertheless, most importantly, to better understand a potential role of ZFP36 proteins in wound healing, the results will have to be reproduced in an in vivo model system, e.g. epidermis-specific knockout animals. Since we could previously demonstrate that ZFP36L1 was a potent regulator of VEGF transcript levels in keratinocytes (Hacker et al., 2010), we also analyzed VEGF production in HaCaT cells after inhibition of ZFP36 expression. As expected, we found that decreased ZFP36 levels also enhanced VEGF production in keratinocytes, indicating that both ZFP36 and ZFP36L1 might con-
Please cite this article in press as: Prenzler, F., et al., Functional analysis of ZFP36 proteins in keratinocytes. Eur. J. Cell Biol. (2016), http://dx.doi.org/10.1016/j.ejcb.2016.04.007
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Please cite this article in press as: Prenzler, F., et al., Functional analysis of ZFP36 proteins in keratinocytes. Eur. J. Cell Biol. (2016), http://dx.doi.org/10.1016/j.ejcb.2016.04.007