Parathyroid hormone-related protein upregulates integrin expression via an intracrine pathway in PC-3 prostate cancer cells

Regulatory Peptides 113 (2003) 17 – 29 www.elsevier.com/locate/regpep

Parathyroid hormone-related protein upregulates integrin expression via an intracrine pathway in PC-3 prostate cancer cells Xiaoli Shen, Miriam Falzon * Department of Pharmacology and Toxicology, and Sealy Center for Molecular Science, University of Texas Medical Branch, 10th and Market Streets, Galveston, TX 77555, USA Received 30 August 2002; received in revised form 25 November 2002; accepted 3 December 2002

Abstract Parathyroid hormone-related protein (PTHrP) is expressed by human prostatic tissue and prostate cancer cell lines, and enhances prostate tumor cell growth both in vivo and in vitro. PTHrP expression also plays a role in the development of bone metastasis, which is a frequent complication in patients with prostate carcinoma. Tumor cell adhesion to extracellular matrix (ECM) components is mediated via integrin subunits, and plays a major role in the invasion and metastasis of tumor cells. We previously showed that PTHrP overexpression increases adhesion of the human prostate cancer cell line PC-3 to the ECM molecules collagen type I, fibronectin, and laminin. Increased adhesion is accompanied by upregulation in the expression of a1, a5, a6, and h4 integrin subunits. We used the same cell line to study the mechanism via which PTHrP upregulates integrin expression. Clonal PC-3 cells were established overexpressing wild-type PTHrP or PTHrP mutated in the nuclear localization sequence (NLS). Mutation of the NLS negated the effects of PTHrP on a1, a5, a6, and h4 integrin expression, indicating that these effects are mediated via an intracrine pathway requiring nuclear localization. Expression of the a2, a3, av, and h1 integrin subunits were comparable in wild-type and NLS-mutated PTHrP transfectants. These findings indicate that PTHrP may play a role in prostate tumor invasion and metastasis by upregulating the expression of specific integrin subunits via an intracrine pathway. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Transfection; Nuclear localization sequence; Integrin; FACS analysis

1. Introduction Parathyroid hormone-related protein (PTHrP) was initially identified through its role in humoral hypercalcemia of malignancy, one of the most frequent paraneoplastic syndromes [1– 4]. More recently, the protein was found to be distributed in most adult and fetal tissues [5,6]. PTHrP can undergo extensive posttranslational processing to generate secretory forms of the protein representing N-terminal, midregion, and C-terminal portions of the protein [7,8]. Each region exhibits unique biological properties, and presumably acts through its own cognate receptor [9,10]. However, only the parathyroid (PTH)/PTHrP (PTH1) receptor that binds PTH, PTHrP, and their N-terminal analogs has been cloned to date [11,12]. PTHrP is not normally present in the circulation [13], indicating that it acts in an autocrine/para* Corresponding author. Tel.: +1-409-772-9638; fax: +1-409-7729642. E-mail address: [email protected] (M. Falzon).

crine, rather than an endocrine, manner. Physiological roles attributed to PTHrP include regulation of cell growth and differentiation, smooth muscle relaxation, promotion of transplacental calcium transport, and cartilage development [7,14 –18]. In addition to effects that are mediated via signal transduction cascades initiated at membrane receptors, PTHrP can also function in an intracrine manner after translocation to the nucleus or nucleolus [19 – 21]. The PTHrP molecule contains a mid-region nuclear localization sequence (NLS, comprised of multibasic clusters in the 88 – 106 region), which resembles the nuclear localization signals found in viral and mammalian transcription factors [19 –22]. Therefore, PTHrP may function locally in an autocrine/paracrine or intracrine manner. Prostate cancer is the most commonly diagnosed cancer in males [23,24] and the second leading cause of cancerrelated deaths in men in the United States [25]. Nearly 70% of prostate cancer patients develop bone lesions, and skeletal metastasis is a major cause of morbidity [26]. The

0167-0115/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. doi:10.1016/S0167-0115(02)00293-8

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prostate is strongly dependent on androgens for normal development and physiological functions. However, additional factors, including growth factors, neuroendocrine peptides, and cytokines, may also play important roles in this organ [27]; one of these factors is PTHrP. Recent studies provide evidence for a role of PTHrP in the development of bone metastases in patients with prostate carcinoma [28,29]. Furthermore, there is increasing evidence that PTHrP plays an important role in mediating local bone breakdown in prostate cancer (reviewed in Ref. [30]). PTHrP has been localized to normal neuroendocrine cells [31], as well as to the glandular epithelium of normal and benign prostatic hyperplasia (BPH) tissues [32]. Cultured epithelial cells derived from normal and BPH tissues synthesize and secrete PTHrP [33], as do immortalized prostate cancer cell lines [34]. The protein’s expression may therefore be associated with both the neuroendocrine and secretory epithelial phenotypes of prostatic cells. PTHrP has also been localized in prostate cancer tissue [35], and is found in higher levels in intraepithelial neoplasia than in normal prostate epithelium [36]. In addition, prostate carcinoma expresses higher levels of PTHrP than does benign prostatic hyperplasia [37]. PTHrP plays both autocrine and intracrine roles in prostate cancer cells [38 – 40]. In the human prostate cancer cell line PC-3, PTHrP exerts a mitogenic role via both autocrine/paracrine and intracrine pathways [38]. We have shown that PTHrP overexpression in PC-3 cells upregulates both the cell surface and mRNA levels of selected integrin subunits [41]. The integrins are a large family of cell adhesion receptors that mediate cell – matrix and cell– cell adhesion. They are transmembrane receptors composed of a and h heterodimer. Both subunits are integral membrane proteins with a large extracellular domain, a transmembrane domain, and a cytoplasmic domain. Different combinations of the a- and h-subunits produce receptors with different ligand specificities [42]. Alterations in integrin composition have been correlated with malignant progression and tumor invasion in vitro and in vivo [43 – 47]. Integrins play a role in prostate cancer metastasis. For example, when prostate cancer cells lose androgen sensitivity after androgen ablation therapy, the tumors become highly invasive and metastatic, an effect that is partly mediated via the a6h4 integrin [48]. Here, we report that the effects of PTHrP on the expression of the a1, a5, a6, and h4 integrin subunits are mediated via an intracrine pathway.

2. Materials and methods 2.1. Materials Synthetic human (h) PTHrP (1-34) was purchased from Bachem (Torrance, CA). Fetal bovine serum and newborn calf serum were obtained from Atlanta Biologicals (Norcross, GA). Tissue culture supplies were purchased from

Life Technologies (Gaithersburg, MD). FuGENEk 6 was obtained from Roche Molecular Biochemicals (Indianapolis, IN). Anti-PTHrP mouse monoclonal antibody was purchased from Oncogene Research Products (Cambridge, MA), and the Tricolor-conjugated goat anti-mouse antibody was purchased from Caltag Laboratories (Burlingame, CA). The R-phycoerythrin (R-PE)-conjugated anti-a1 (CD49a; clone SR84), anti-a2 (CD49b; clone 12F1-H6), anti-a3 (CD49c; clone C3 II.1), anti-a5 (CD-49e; clone IIA), antia6 (CD49f; clone GoH3), anti-av/h3 (CD51/61; clone 23C6), anti-h1 (CD29; clone MAR4), and anti-h4 (clone 439-9B) antibodies, as well as the isotype controls [mouse IgG1, n-PE (clone MOPC-1), mouse IgG2a, n-PE (clone G155-178) and rat IgG2a, n-PE (clone R35-95)] were obtained from BD PharMingen (San Diego, CA). The multiprobe template set (human ITG-1) for analysis of integrin-related genes was also purchased from BD PharMingen. 2.2. Plasmid constructs A cDNA encoding human PTHrP (obtained from Genentech, South San Francisco, CA) was digested with EcoRI and HindIII and subcloned in the sense orientation into the expression vector pcDNA3.1(+) (Invitrogen, San Diego, CA) [(+) indicates the same orientation of the multiple cloning site within the vector as the direction of transcription from the T7 promoter]. This construct was used to prepare PTHrP cDNAs mutated in the NLS, using a Transformer Site-Directed Mutagenesis Kit (Clontech Laboratories, Palo Alto, CA). The cDNAs encoded either a single deletion (elimination of residues 88-91 or 102-106), or a double deletion (elimination of residues 88-91 and 102106). Mutations were confirmed by DNA sequencing. These constructs, as well as the empty vector control pcDNA 3.1(+), were transfected into PC-3 cells using the FuGENEk 6 Transfection Reagent (Roche Molecular Biochemicals). The DNA template used to prepare the probe for Northern blot analysis was a 231-bp cloned DNA fragment spanning exons 3 and 4 of the human PTHrP gene [49,50]. 2.3. Cell culture and stable transfection PC-3 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and grown at 37 jC in a humidified 95% O2 –5% CO2 atmosphere in Ham’s F-12 medium supplemented with 7% fetal bovine serum (FBS; Atlanta Biologicals) and L-glutamine (Life Technologies). PC-3 cells were stably transfected using the FuGENEk 6 Transfection Reagent, according to the manufacturer’s specifications. Two days after transfection, 600 Ag/ml G418 (Geneticin; Life Technologies) was added and resistant clones were selected. Single clones of stably transfected cells, isolated by limiting dilution in 96-well plates, were

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transferred to individual flasks and cultured in medium containing 300 Ag/ml G418. Individual clones were tested for PTHrP production using an immunoradiometric assay (described below), and endogenous and transfected messenger RNAs (mRNAs) were detected by Northern blot analysis (also described below). To measure the effects of added hPTHrP (1-34) on integrin expression, PC-3 cells were plated into T75 flasks in medium containing 7% FBS. In some experiments, cells were transferred to medium containing 2% FBS after 24 h. After a further 24 h, cells were treated with the indicated concentrations of PTHrP (1-34), prepared in 10 mM acetic acid (at 10 4 M). Control cells received an equivalent volume of vehicle (10 mM acetic acid). Integrin expression was measured after 1– 5 days of treatment. When cells were treated with peptide for 5 days, the growth medium was replaced with fresh peptide-containing medium after 3 days. Integrin mRNA and cell surface expression levels were measured by the ribonuclease protection assay (RPA) and FACS analysis, respectively, as described below. 2.4. Northern blot analysis Total RNA was isolated using RNA STAT-60 (Tel-Test ‘B’, Friendswood, TX). RNA gel electrophoresis was performed as previously described [49], using 25 Ag of total RNA. The RNA was then blotted onto nitrocellulose (Schleicher and Schuell, Keene, NH) by capillary action. Probes for hybridization were labeled by the asymmetric polymerase chain reaction (PCR), using 32P-dATP [51]. The PCR template was a 231-bp cloned cDNA fragment spanning exons 3 and 4 of the human PTHrP gene [49,50]. To detect the endogenous and transfected sense PTHrP mRNA, an antisense probe was prepared using the downstream primer from exon 4 of the human PTHrP gene (5VGTTAGGGGACACCTCCGAGGT-3V). The blots were prehybridized for 30 min and hybridized for 2 h in Expresshyb (Clontech Laboratories) at 65 jC. After hybridization, the blots were washed twice in 2  SSC (1  SSC is 0.15 M NaCl plus 0.15 M sodium citrate), 0.05% SDS for 15 min at room temperature, and then twice in 0.1  SSC, 0.1% SDS at 50 jC for 30 min. The washed membranes were exposed to Kodak X-Omat film (Eastman Kodak, Rochester, NY) at 80 jC with intensifying screens for 48 h. The ethidium bromide-stained RNA on the nitrocellulose membrane was photographed immediately after transfer. The 18S ribosomal RNA signal was used to provide a reference to normalize for equal RNA loading and transfer. The intensities of the bands representing PTHrP and 18S ribosomal RNA were evaluated using the Sigmagel program (Jandel Scientific, San Rafael, CA). 2.5. Immunoassay for secreted PTHrP The amount of PTHrP secreted into the culture medium was measured using an immunoradiometric ‘sandwich’

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assay (Nichols Institute, San Juan Capistrano, CA), as previously described [49]. Single clones of transfected and control (untransfected) cells were plated in 24-well dishes (5  104 cells/well), and the medium was replaced after 24 h. Conditioned medium was collected after a further 4 days and frozen at 80 jC for future use; the cell number was determined using a Coulter counter. Unconditioned medium (never exposed to cells) served as a negative control. The assay was carried out per the manufacturer’s specifications. The detection limit of the assay is 0.7 pmol/l [52]. 2.6. Ribonuclease protection assay Total RNA was isolated using RNA STAT-60 (Tel-Test ‘B’). To determine the levels of expression of the integrin family of genes, the multiprobe template set human ITG-1 (BD PharMingen) was used. Antisense RNA probes labeled with 32P-uridine 5V-triphosphate were prepared from this template set, and 4 Ag of total RNA from each sample was hybridized with the antisense probes according to the manufacturer’s specifications. After digestion of unbound RNA and ethanol precipitation of RNA/probe complexes, samples were analyzed on a 5% polyacrylamide/8 M urea gel. The gels were dried on 3M filter paper (Whatman, Maidstone, UK) and then exposed to X-ray film at 80 jC for 16 h. The films were analyzed by densitometry and quantified using Applied Imaging Lynx 5000 software (Biological Vision, San Mateo, CA). The densities of individual integrin bands were normalized to glyceraldehyde-phosphate dehydrogenase (GAPDH) and the 60S ribosomal protein L32 to correct for variations in loading of gels. 2.7. Cell synchronization Cells were plated in T75 flasks in medium containing 7% FBS. At 50% confluence, the medium was replaced with fresh medium containing 10 Ag/ml aphidicolin. After 24 h, the medium was removed and the cell monolayer was washed twice with phosphate-buffered saline (PBS). The cells were then incubated for a further 24 h in fresh medium (in the absence of aphidicolin). After 24 h, the cells were trypsinized, and integrin expression was measured as described below. 2.8. Flow cytometry The cell surface expression of integrin subunits was measured in aphidicolin synchronized (described above) and unsynchronized cells by staining with R-PE-labeled antibodies to the a1, a2, a3, a5, a6, av/h3, h1 or h4 integrin subunits. For these experiments, 2  105 cells were trypsinized, washed with PBS, and incubated with the specific antibody for 20 min. After two washes with PBS containing 2% newborn calf serum (NCS) and 0.02%

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sodium azide, the mean fluorescence intensity was measured on a FACS Scan flow cytometer (Becton Dickinson, Franklin Lakes, NJ). Viable cells were electronically gated using forward and side scatter parameters. For the a1 and a5 integrins, data are presented as the percentage of cells (wild-type or NLS-mutated PTHrP-overexpressing, or empty vector controls) expressing the respective integrin. Because all cells were positive for a6 and h4 integrin subunits, the data are presented as a comparison between the log fluorescence intensity of wild-type PTHrP-overexpressing vs. NLS-mutated PTHrP-overexpressing cells, rather than the percentage of a6- or h4-positive cells. Double staining with anti-integrin and anti-PTHrP antibodies was carried out as follows. Cells (0.2  106) were trypsinized, washed with PBS containing 2% NCS and 0.02% sodium azide, and incubated on ice for 20 min with antibodies to the a1, a5, a6, or h4 integrin subunits. After two washes with PBS, the cells were fixed for 60 min on ice with 2% paraformaldehyde, and permeabilized by incubation with 0.2% Saponin, 2% NCS, 1 mM CaCl2, and 1 mM MgCl2 in PBS on ice for 1 h. Nonfat dry milk (5%) was included to block adventitious binding sites. The cells were then incubated with anti-PTHrP antibody or mouse IgG2a (isotype control) for 1 h, washed twice with PBS containing 0.1% Saponin, 2% NCS and 0.02% sodium azide, and incubated with goat anti-mouse IgG (secondary antibody; TRI-color conjugated) for 1 h. After two washes with PBS containing 0.1% Saponin, 2% NCS and 0.02% sodium azide, and one wash with PBS containing 2% NCS and 0.02% sodium azide, expression of the cell surface (integrins) and intracellular (PTHrP) antigens was determined by two-color flow cytometry analysis on a FACS Scan flow cytometer, as described above.

cDNA constructs mutated at the NLS. Transfected wildtype and NLS-mutated sense PTHrP mRNAs were detected by Northern blot analysis. Transfected sense PTHrP mRNA was only detected in PC-3 cells transfected with the sense wild-type or NLS mutant PTHrP expression constructs ( f 1.1-kb transcript; Fig. 1), but not in untransfected or empty vector-transfected cells (Fig. 1). [Longer exposure of blots showed the presence of the endogenous 1.5-kb transcript in both transfected and untransfected cells (data not shown).] Notably, transfection of PC-3 cells with the wild-type or NLS-mutated PTHrP cDNA constructs, or the empty vector did not change cell morphology, compared to the parent (untransfected) cells (data not shown). PTHrP secretion was measured by immunoassay. The amount of PTHrP secreted by control cells was 2.2 F 0.04 fmoles/105 cells. PTHrP secretion from the empty vectortransfected cells was not significantly different from that by untransfected cells (Fig. 2). Transfection with the wild-type PTHrP construct produced a significant increase in PTHrP production compared to empty vector-transfected and untransfected cells (Fig. 2). Similarly, each of the three NLS-mutated PTHrP transfectants (D88-91, D102-106, and D88-91 + D102-106) secreted significantly higher PTHrP levels (Fig. 2). During the process of cloning, secretion from a minimum of four clones for wild-type PTHrP and each of the deletions was tested. These clones secreted between 30 and 90 fmoles PTHrP per 105 cells. Two clones for wild-type PTHrP and each of the NLS mutants (D88-91, D102-106 and D88-91 + D102 – 106) were chosen for further experiments to examine whether deletion of the NLS abolishes the PTHrP-induced upregulation in integrin expression.

2.9. Statistics Numerical data are presented as the mean F S.E.M. The data were analyzed by ANOVA followed by a Bonferroni post-test to determine the statistical significance of differences. All statistical analyses were performed using Instat Software (GraphPad Software, San Diego, CA). Flow cytometry data were analyzed using the CellQuest program (Becton Dickinson). P < 0.05 was considered significant.

3. Results 3.1. Establishment and characterization of cell lines overexpressing NLS-mutated PTHrP We have previously shown that transfection of PC-3 cells with a sense PTHrP construct upregulates expression of the a1, a5, a6, and h4 integrin subunits [41]. In order to determine whether these effects of PTHrP in PC-3 cells require an intact NLS, cells were transfected with PTHrP

Fig. 1. Characterization of PC-3 cells overexpressing wild-type or NLSmutated PTHrP. Northern blot analysis of total RNA was carried out to detect transfected PTHrP transcripts from parent (untransfected) cells (P), and from each of two individual clonal cell lines transfected with empty vector (V1 and V2), with a vector expressing wild-type sense PTHrP (SN1 and SN2) or with sense vectors expressing NLS-mutated PTHrP mRNA (D1,1 and D1,2 = D88-91; D2,1 and D2,2 = D102-106; and DD1 and DD2 = D88-91 + D102-106). The probe was prepared by asymmetric PCR of a cDNA fragment spanning exons 3 and 4 of the human PTHrP gene as described in Materials and methods. The position of the 18S ribosomal bands is indicated as is the position of the endogenous PTHrP transcript (visible on longer exposure of the autoradiographs; dashed arrow). Top panel, PTHrP mRNA; bottom panel, 18S RNA.

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Fig. 2. PTHrP secretion by wild-type and NLS-mutated PTHrP transfectants. Conditioned medium from PC-3 cells transfected with a vector expressing wild-type or NLS-mutated PTHrP was collected after 4 days in culture; secreted PTHrP was measured by immunoradiometric assay. P = parent (untransfected) cells; V = empty vector-transfected cells; SN = cells expressing wild-type PTHrP; D1, D2, and DD, cells expressing D8891, D102-106, and D88-91 + D102-106 PTHrP, respectively. Each bar, representing an independent clone, is the mean F S.E.M. of four wells, obtained after subtracting the background value, represented by unconditioned medium (not exposed to cells). *Significantly different from parent cells ( P < 0.001).

3.2. Mutation of the NLS negates the upregulatory effects of PTHrP on the expression of the a1, a5, a6 and b4 integrin subunits The cell surface expression of the a1, a2, a3, a5, av/ h3, a6, h1, and h4 integrin subunits in PC-3 cells overexpressing wild-type or NLS-mutated PTHrP, and in control (empty vector-transfected) cells, was determined by FACS analysis. There was no detectable expression of the av/h3 integrin subunit in wild-type or NLS-mutated PTHrP-overexpressing cells, or in control cells (data not shown). The a2, a3, and h1 subunits were highly expressed on wild-type and NLS-mutated PTHrP-overexpressing cells, as well as on control cells; FACS analysis did not demonstrate a change in the expression of these integrin subunits in PTHrP-overexpressing vs. control cells (data not shown). Wild-type (sense) PTHrP enhanced the expression of the a1 and a5 integrin subunits (Fig. 3). Thus, a significantly higher percentage of wild-type PTHrP-overexpressing cells showed cell surface expression of the a1 and a5 integrin subunits, compared to control (empty vector-transfected) cells (Fig. 3A –D). In contrast, the a1 and a5 integrins were expressed in a significantly lower percentage of cells transfected with the D88-91, D102-106, or D88-91 + D102106 PTHrP cDNA, compared to cells transfected with the cDNA coding for the wild-type protein (Fig. 3A – D).

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Unlike a1 and a5, the a6 and h4 integrin subunits were expressed on the cell surface of PTHrP-overexpressing and empty vector-transfected PC-3 cells. However, the expression of these two integrins was significantly higher in cells overexpressing wild-type PTHrP than in control cells (Fig. 4A – D), indicating upregulation by PTHrP. Again, deletion of the NLS negated the upregulatory effects of PTHrP on a6 and h4 integrin expression (Fig. 4A – D). In Fig. 4A,C, the scan with isotype control antibody was generated using wild-type PTHrP-overexpressing cells. Overlapping scans were obtained when the NLS-mutated PTHrP and empty vector transfectants were incubated with isotype control antibody (data not shown). These data indicate that, on incubation with antibodies to the a6 or h4 integrin subunits, the shifted scan in wild-type PTHrP-overexpressing vs. NLS-mutated PTHrP-overexpressing and empty vector transfectants is truly representative of higher cell surface expression of the a6 (Fig. 4A) and h4 (Fig. 4C) integrin subunits in wild-type PTHrP overexpressing cells. These results, as well as those presented in Fig. 3, also indicate that the presence of an intact NLS is necessary for PTHrP to exert its effects on integrin subunit expression. There was no significant difference in the expression of any of the integrins tested between parental (untransfected) and empty vector-transfected cells (data not shown). The same experiments were carried out using aphidicolin-synchronized cells. A very similar integrin expression profile was obtained, with wild-type PTHrP-overexpressing cells showing significantly higher cell surface expression of the a1, a5, a6, and h4 integrins than NLSmutated PTHrP-overexpressing and control cells (data not shown). A summary tabulating the relative expression of the a1, a5, a6, and h4 integrins in the PC-3 cell clones overexpressing wild-type and NLS-mutated PTHrP in presented in Table 1. 3.3. The NLS-dependent effects of PTHrP on integrin expression are mediated through a transcriptional pathway RNase protection assays were carried out to determine whether the PTHrP-mediated effects on integrin subunit expression occur at the transcriptional level. For this purpose, a multiprobe system capable of simultaneous analysis of multiple members of the integrin family was used. Analysis of the housekeeping genes GAPDH and L32 ensured the integrity of the RNA and gel loading efficiency. Gel electrophoresis of the labeled multi-RNA probe showed comparable labeling efficiencies of all the components (data not shown). As shown in Fig. 5A, PC-3 cells express very low levels of the a1, a4, a5, a7, a8, and a9 integrin subunits. The a3 and h1 subunits are the predominant integrins expressed by these cells, while expression of the a2, av, and a6 integrins is intermediate. Overexpression of wild-type PTHrP resulted in a significantly higher expression of the a1, a5, and a6 integrin

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Fig. 3. Cell surface expression of a1 (A and B) and a5 (C and D) integrin subunits in PC-3 cells overexpressing wild-type or NLS-mutated PTHrP. Cells were stained with phycoerythrin (PE)-conjugated antibodies against a1 or a5 integrins and analyzed by FACS analysis as described in Materials and methods. SN = wild-type PTHrP transfectants; V = empty vector transfectants; D1 = D88-91 PTHrP; D2 = D102-106 PTHrP; DD = D88-91 + D102-106 PTHrP. (A and C) The percentage of a1- (A) or a5- (C) positive cells is shown. The dashed line represents the scan obtained with the isotype control antibody. Positive cells were delineated as those cells whose log fluorescence intensity was equal to or greater than that of the isotype control antibody-stained cells (shown as Mark 1 or M1). (B and D) Each bar is the mean F S.E.M. of three independent experiments. Where no error bar is shown, the S.E.M. is smaller than the bar line. *Significantly different from all other values ( P < 0.001).

subunits vs. empty vector-transfected cells (Fig. 5A,B). However, expression of the a1 and a5 integrins was comparable in the NLS-mutated PTHrP and empty vector transfectants (Fig. 5A,B). Expression of the a6 integrin was significantly lower in the NLS-mutated PTHrP clones than in the clones overexpressing wild-type PTHrP (Fig. 5A,B). However, the single NLS mutants (D88-91 or D102-106) still expressed higher a6 integrin levels than the empty

vector transfectants (Fig. 5A,B). These results complement the FACS analysis data (Figs. 3 and 4). Wild-type or NLSmutated PTHrP produced no significant change in expression of the a2, a4, a7, a8, or a9 subunits (Fig. 5A,B and data not shown). However, in contrast to the flow cytometry data, there was increased expression of the a3 and h1 integrins in wild-type PTHrP-overexpressing vs. control cells (Fig. 5A,B). Mutation of the NLS negated this effect

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Fig. 4. Cell surface expression of a6 (A and B) and h4 (C and D) integrin subunits in PC-3 cells overexpressing wild-type or NLS-mutated PTHrP. Cells were stained with phycoerythrin (PE)-conjugated antibodies against a6 or h4 integrins and analyzed by FACS analysis as described in Materials and methods. SN = wild-type PTHrP transfectants; V = empty vector transfectants; D1 = D88-91 PTHrP; D2 = D102-106 PTHrP; DD = D88-91 + D102-106 PTHrP. (A and C) The dashed line represents the scan obtained with the isotype control antibody. Since both PTHrP-overexpressing and control cells express a6 and h4 integrin subunits, data are presented as the percentage of cells whose log fluorescence intensity is greater than or equal to Mark 1 (M1) (channels 246 and 319 for a6 and h4 integrins, respectively). (B and D) Each bar is the mean F S.E.M. of three independent experiments. Where no error bar is shown, the S.E.M. is smaller than the bar line. *Significantly different from all other values ( P < 0.001).

(Fig. 5A,B). Expression of the av subunit was elevated f 2-fold in the single PTHrP mutants (D88-91 or D102106), compared to the wild-type-PTHrP-overexpressing Table 1 Cell surface expression of the a1, a5, a6, and h4 integrins in PC-3 cells overexpressing wild-type or NLS-mutated PTHrP Clone a1 Positive

SN1 SN2 V1 V2 D1,1 D1,2 D2,1 D2,2 DD1 DD2

a5 Positive

a6 High

h4 High

Mean

F Mean S.E.M.

F Mean S.E.M.

F Mean S.E.M.

F S.E.M.

74.5* 81.5* 18.0 24.3 25.4 10.5 42.5 51.5 5.4 5.9

2.7 2.1 3.4 2.2 2.3 3.1 0.8 1.4 0.4 0.3

0.7 1.1 1.4 0.1 4.3 1.4 5.2 5.8 2.1 2.8

5.4 4.4 0.8 1.2 3.1 4.7 4.4 6.4 1.4 1.5

2.6 2.2 2.7 4.2 3.2 6.4 5.2 6.8 1.5 1.7

86.5* 78.5* 13.2 17.1 23.5 20.5 53.5 46.5 17.1 20.8

71.7* 80.1* 17.4 21.9 30.7 21.3 52.5 40.3 29.0 18.0

clones (Fig. 5A). These results were not evident by FACS, where av expression was below the levels of detection. The results presented here were obtained using GAPDH as an internal control. Essentially the same results were obtained when L32 was used as the internal control (data not shown). The h4 integrin is not part of the array, so its expression was not tested by this RPA system.

74.0* 80.7* 19.7 19.6 31.0 26.6 54.5 51.5 28.6 22.1

Cells were stained with phycoerythrin-conjugated antibodies against these integrins and analyzed by FACS analysis as described in Materials and methods. SN = wild-type PTHrP transfectants; V = empty vector transfectants; D1 = D88-91; D2 = D102-106; DD = D88-91 + D102-106. Each value is the mean F S.E.M. of three independent experiments. Values for integrins a1 and a5 represent the percentage of cells expressing these integrins as determined in Fig. 3; values for integrins a6 and h4 represent the percentage of cells expressing high levels of these integrins as determined in Fig. 4. * Significantly different from all other values respective ( P < 0.001).

3.4. Direct correlation between intracellular PTHrP levels and cell surface integrin expression We used two-color flow cytometry analysis to examine whether there is a correlation between intracellular (cytoplasmic plus nuclear) PTHrP levels and expression of the a1, a5, a6, and h4 integrin subunits. We have previously shown by immunofluorescence that PC-3 cells overexpressing wild-type PTHrP show strong cytoplasmic and nuclear staining. In contrast, NLS-mutated PTHrP transfectants show very low nuclear staining, but strong cytoplasmic staining is still present [38]. Both wild-type and NLSmutated PTHrP transfectants showed a range of intracellular PTHrP levels, as indicated by the scatter in the two left quadrants (Fig. 6A). This may reflect different stages within the cell cycle [20,21,53 – 55]. In fact, Gujral et al. [55] report that nuclear staining of PTHrP in PC-3 cells varied in intensity and homogeneity, and attribute this to the cells being in the various phases of the cell cycle. A

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Fig. 5. Analysis of integrin-related genes in PC-3 cells overexpressing wild-type or NLS-mutated PTHrP. (A) Total RNA was isolated from each sample and analyzed using a multi-probe RNase protection assay as described in Materials and methods. SN = sense transfectants; V = empty vector transfectants; D1 = D88-91 PTHrP; D2 = D102-106 PTHrP; DD = D88-91 + D102-106 PTHrP. (B) Each bar is the mean F S.E.M. of three independent experiments for each of two independent clones obtained after densitometric analysis and normalization to GAPDH. The V value is set arbitrarily at 1.0. Empty bar = V; upward stippled bar = SN; cross-hatched bar = D1; downward stippled bar = D2; horizontal lined bar = DD. *Significantly different from respective V value ( P < 0.001).

significantly greater percentage of wild-type PTHrP transfectants expressed high levels of both PTHrP and a1, a5, a6, or h4 integrins, compared to the NLS-mutated PTHrP transfectants (Fig. 6A,B). In Fig. 6, data are shown for the wild-type (SN) and D88-91 + D102-106 (DD) PTHrP transfectants. Similar results were obtained for the single (D88-

91 or D102-106) PTHrP mutants (data not shown). In contrast, the majority of NLS-mutated PTHrP transfectants had low cell surface expression of the a1, a5, a6, or h4 integrins, even in the presence of high intracellular PTHrP concentrations (Fig. 6A, left quadrants, Fig. 6B; data not shown).

X. Shen, M. Falzon / Regulatory Peptides 113 (2003) 17–29

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Fig. 6. Correlation between cell surface integrin expression and intracellular PTHrP levels in PC-3 cells overexpressing wild-type or NLS-mutated PTHrP. Cells were stained with phycoerythrin (PE)-conjugated antibodies against a1, a5, a6, or h4 integrins, then fixed, permeabilized, stained with anti-PTHrP antibody, and analyzed by two-color FACS analysis as described in Materials and methods. SN = wild-type PTHrP transfectants; DD = D88-91 + D102-106 PTHrP transfectants. (A) Data are presented as PTHrP (vertical axis) vs. integrin (horizontal axis) expression. For a1 and a5 integrins, placement of quadrant markers on the x-axis (vertical lines) was determined according to the isotype control antibody staining. For these integrins, the vertical quadrants distinguish between positive and negative a1 or a5 expression. For a6 and h4 integrins, the quadrant markers on the x-axis separate cells into high- and low-expressing populations. Quadrant markers on the y-axis (horizontal lines) distinguish between high and low PTHrP expression. (B) The dotted bars show the percentage of integrin-positive (a1 and a5) or integrin high-expressing (a6 or h4) cells among the high PTHrP-expressing cells. The stippled bars show the percentage of integrin-positive (a1 and a5) or integrin high-expressing (a6 and h4) cells among the low PTHrP-expressing cells. Each bar is the mean F S.E.M. of four independent experiments (two experiments for each of two individual clones). Where no error bar is shown, the S.E.M. is smaller than the bar line. *Significantly different from all other values ( P < 0.001).

3.5. Exogenously added hPTHrP (1-34) has no effect on PC-3 integrin expression We examined the cell surface integrin expression of parental (untransfected) PC-3 cells to which hPTHrP (134) had been added. This PTHrP moiety interacts with the cell surface PTH/PTHrP receptor, but does not access the nucleus/nucleolus because it lacks an NLS [20,56]. Addition of hPTHrP (1-34) (10 7 M) for 72 h to cells cultured in the presence of 2% FBS (Fig. 7) or 7% FBS (data not shown)

had no significant effect on the cell surface expression of the a1, a5, a6, or h1 integrin subunits. Addition of hPTHrP (134) had no effect on the autofluorescence (background fluorescence) of the cells, compared to vehicle-treated (10 mM acetic acid) or untreated cells (data not shown). Similar results were obtained after a 24-, 48-, or 120-h treatment (data not shown). Under these conditions, hPTHrP (1-34) has been shown to stimulate PC-3 cell proliferation [38,40]. These results were confirmed using the multi-probe RPA assay. Addition of hPTHrP (1-34) (10 7 – 10 9 M) for

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X. Shen, M. Falzon / Regulatory Peptides 113 (2003) 17–29

Fig. 7. Effects of exogenously added hPTHrP (1-34) on cell surface integrin expression of parental PC-3 cells. Cells were plated in 7% FBS, then transferred to 2% FBS after 24 h. hPTHrP (1-34) (10 7 M) was added after a further 24 h. Expression of a1, a5, a6, and h4 integrin subunits was determined after 72 h by FACS analysis as described in Materials and methods. (A) Dashed line = scan with isotype control antibody; thin line = scan with vehicle-treated (10 mM acetic acid) cells; thick line = scan with cells treated with hPTHrP(1-34). (B) MFI = mean fluorescence intensity. Each bar is the mean F S.E.M. of four independent experiments. Where no error bar is shown, the S.E.M. is smaller than the bar line. Empty bar = vehicle control; stippled bar = hPTHrP (1-34).

intervals of 24 to 120 h had no effect on mRNA levels of the a1, a5, and a6 integrin subunits (data not shown).

4. Discussion In this study, we examined the pathway(s) via which PTHrP regulates integrin expression, using the human prostate cancer cell line PC-3 as a model. This cell line provides a good system to study both the autocrine/paracrine and intracrine effects of PTHrP. Thus, PC-3 cells express functional PTH/PTHrP receptors [38]. When wild-type PTHrP is overexpressed in PC-3 cells, it accumulates in the nucleus. In contrast, nuclear localization is virtually absent in PC-3 cells overexpressing NLS-mutated PTHrP [38]. Our main findings are that PTHrP upregulates integrin expression via an intracrine pathway. Notably, we show a direct correlation between wild-type PTHrP expression and cell surface levels of the a1, a5, a6, and h4 integrin subunits. PTHrP increases PC-3 cell proliferation via both autocrine/paracrine and intracrine pathways [38]. Thus, we and others [38,40] have shown that addition of PTHrP (1-34) to

PC-3 cells cultured in 2% or 7% FBS increases both cell number and [3H]thymidine incorporation, indicating that this PTHrP moiety exerts a biological effect in PC-3 cells. In the present study, PTHrP (1-34) did not modulate mRNA levels or the cell surface expression of any of the integrin subunits tested. PTHrP (1-34) was used because this peptide lacks an NLS, and is therefore not targeted to the nucleus [56]. Longer peptides, such as PTHrP (1-86), have been localized to the nucleus, possibly through interaction with other, still unidentified, targeting proteins, even without a canonical NLS [55]. It therefore appears that the effects of PTHrP on integrin expression in PC-3 cells are independent of the PTH/PTHrP receptor. However, it is still possible that other, non-N-terminal PTHrP analogues, functioning through distinct pathway(s), may exert an autocrine/paracrine effect on integrin expression. Endogenous overexpression of wild-type PTHrP in PC-3 cells selectively increased expression of the a1, a5, a6, and h4 integrin subunits. The nuclear effects of PTHrP can be achieved via protein-receptor endocytosis of ligand –PTH/ PTHrP receptor complexes [57]. PTH/PTHrP receptor-independent endocytosis has also been suggested [56]. Both receptor-dependent and-independent trafficking require the presence of an intact NLS [56]. In order to confirm the involvement of the nuclear mechanism of action, we measured integrin expression in PC-3 cell clones overexpressing NLS-mutated PTHrP, an approach often used to address the mechanism of action of this protein [38,39,53,58 – 61]. Our results showed that deleting either multibasic cluster (D8891, D102-106, or D88-91 + D102-106) in the NLS of PTHrP negated the effects on integrin expression at both the mRNA and cell surface level. We also showed a direct correlation between wild-type PTHrP expression and cell surface levels of the a1, a5, a6, and h4 integrin subunits. Since both wildtype and NLS-mutated PTHrP clones exhibit high cytoplasmic levels of PTHrP, but only wild-type PTHrP accumulates in the nucleus [38], the effects of PTHrP on integrin expression must be mediated via an intracrine pathway. The single NLS-mutated PTHrP clones target two different regions of PTHrP. Amino acids 88-91 are recognized by h-importin [57], and it appears that deletion of this region contributes most significantly to the intracrine effects of PTHrP on integrin expression. In contrast, cell surface expression of the a1, a5, a6, and h4 integrins was higher in the D102-106 PTHrP clones, such that integrin levels in the D102-106 mutants were intermediate between the wildtype PTHrP-overexpressing clones and the clones lacking the 88-91 sequence. It therefore appears that the h-importin pathway, requiring the presence of amino acids 88-91, is primarily involved in integrin subunit regulation by PTHrP in PC-3 cells. These results contrast with our previous observations that the presence of amino acids 102-106 contributes more to the proliferative effects of PTHrP in PC-3 cells, as measured by [3H]thymidine incorporation [38]. We also reported that PTHrP shows both diffuse nuclear staining and distinct nucleolar localization in this

X. Shen, M. Falzon / Regulatory Peptides 113 (2003) 17–29

cell line [38]. A possible scenario may be that the two independent pathways for importing PTHrP to the nucleus (mediated by amino acids 88-91 or 102-106) may localize the protein to the nucleus vs. the nucleolus, and that the effects on cell proliferation vs. integrin subunit expression may be dependent on subnuclear localization. The integrins mediate both cell – matrix and cell– cell adhesion. The a1h1 integrins mediate attachment to collagen type 1, a5h1 is involved in fibronectin adhesion, and both a6h1 and a6h4 integrins are involved in attachment to laminin [42]. These extracellular matrix (ECM) components are expressed in the interstitial matrix (collagen type I) and basement membrane (fibronectin and laminin). We have previously shown that PTHrP overexpression in PC-3 cells increases their adhesion to these three ECM molecules [41]. In addition, overexpression of the a5 integrin has been shown to correlate with the resistance of cancer cells to undergo apoptosis. Thus, the a5h1 integrin protects intestinal epithelial cells against apoptosis caused by growth factor deprivation; the mechanism involves selective activation of the phosphatidylinositol-3-kinase (PI-3-kinase) pathway [62]. PTHrP overexpression also protects PC-3 cells from apoptosis (Shen and Falzon, unpublished data); enhanced survival may be mediated, at least partially, via the PTHrPmediated upregulation of the a5 integrin. Expression of the a6 integrin subunit has been shown to correlate closely with the invasive potential of several cancer cells [63]. The a6 subunit can associate with the h1 subunit to form a6h1; high levels of this heterodimer facilitate the attachment of tumor cells to the basement membrane, and thereby the transversion of these cells, a prerequisite for cancer cells to gain access to other tissue compartments and metastasize to bone [64 – 67]. PTHrP overexpression also upregulates the cell surface expression of the h4 subunit; this integrin associates exclusively with the a6 subunit to form the a6h4 heterodimer [68]. Expression of the a6h4 integrin has been linked to the aggressiveness of several tumor cell lines (reviewed in Ref. [63]). For example, in prostate cancer cell lines, androgen receptor expression suppresses a6h4 integrin expression and the invasive phenotype, suggesting that the loss of androgen receptor expression, a common feature of aggressive prostate tumors, stimulates a6h4 expression and a6h4-mediated invasion [48]. Thus, upregulation of integrin subunit expression by PTHrP may have important implications for bone metastasis. PTHrP expression increases as cells metastasize to bone [69,70]; this high PTHrP concentration may upregulate integrin expression in situ, thereby further promoting cancer cell adhesion and localization in a spatially regulated manner. As demonstrated by flow cytometry, PTHrP overexpression in PC-3 cells selectively upregulated the cell surface expression of the a1, but not of the a2, a3, or h1 subunits. Conversely, in addition to a1 mRNA, a3 and h1 mRNA levels were also upregulated by PTHrP, as determined by RPA. It may be that since control PC-3 cells exhibit high cell surface expression of the a3 and h1 integrins (data not

27

shown, [48,71]) further induction of the mRNA levels of these two integrins by PTHrP does not necessarily lead to elevated cell surface expression. In conclusion, the results presented here demonstrate that endogenously overexpressed PTHrP upregulates expression of the a1, a5, a6, and h4 integrin subunits. The presence of an intact NLS is required for these effects. The PTHrPmediated increase in integrin expression may promote prostate cancer cell adhesion, migration and invasion, thereby facilitating colonization of the bone matrix by prostate cancer cells, and thus metastasis.

Acknowledgements We wish to thank Dr. Rolf Konig for his advice with FACS analysis, and Drs. D. Konkel and P.K. Seitz, for their critical reading of the manuscript. This work was supported by NIH Grant CA83940.

References [1] Burtis WJ, Wu T, Bunch C, Wysolmerski JJ, Insogna KL, Weir EC, et al. Identification of a novel 17,000 dalton parathyroid hormone-like adenylate cyclase-stimulating protein from a tumor associated with humoral hypercalcemia of malignancy. J Biol Chem 1987;262:7151 – 6. [2] Moseley JM, Kubota M, Diefenbach-Jagger H, Wettenhall REH, Kemp BE, Suva LJ, et al. Parathyroid hormone-related protein purified from a human lung cancer cell line. Proc Natl Acad Sci U S A 1987;84:5048 – 52. [3] Suva LJ, Winslow GA, Wettenhall REH, Hammonds RG, Moseley JM, Diefenbach-Jagger H, et al. A parathyroid hormone-related protein implicated in malignant hypercalcemia: cloning and expression. Science 1987;237:893 – 6. [4] Strewler GI. Mechanisms of disease: the physiology of parathyroid hormone-related protein. N Engl J Med 2000;342:177 – 85. [5] Philbrick WM, Wysolmerski JJ, Galbraith S, Holt E, Orloff JJ, Yang KH, et al. Defining the role of parathyroid hormone-related protein in normal physiology. Physiol Rev 1996;76:127 – 73. [6] Yang KH, Stewart AF. The PTH-related protein gene and protein products. In: Bilezikian JP, Riasz L, Rodan G, editors. Principles of bone biology. San Diego: Academic; 1996. p. 347 – 76. [7] Orloff JJ, Reddy Y, de Papp AE, Yang KH, Soifer NE, Stewart AF. Parathyroid hormone-related protein as a prohormone posttranslational processing and receptor intersections. Endocrine Rev 1994;15: 40 – 60. [8] Wu T, Vasavada R, Yang K, Massfelder T, Ganz M, Abbas SK, et al. Structural and physiologic characterization of the mid-region secretory species of parathyroid hormone-related protein. J Biol Chem 1996;271:24371 – 81. [9] Orloff JJ, Kats Y, Urena P, Schipani E, Vasavada RC, Philbrick WM, et al. Further evidence for a novel receptor for amino-terminal parathyroid hormone-related protein on keratinocytes and squamous carcinoma cell line. Endocrinology 1995;136:3016 – 23. [10] Mannstadt M, Ju¨ppner H, Gardella TJ. Receptors for PTH and PTHrP. Am J Physiol 1999;277:F665 – 75. [11] Ju¨ppner H, Abou-Samra A-B, Freeman M, Kong XF, Schipani E, Richards J, et al. A G protein-linked receptor for parathyroid hormone and parathyroid hormone-related peptide. Science 1991;254:1024 – 6. [12] Abou-Samra A-B, Ju¨ppner H, Force T, Freeman MW, Kong X-F, Schipani E, et al. Expression cloning of a common receptor for parathyroid hormone and parathyroid hormone-related peptide from rat

28

[13] [14]

[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22] [23] [24] [25]

[26] [27] [28]

[29] [30] [31]

[32]

[33]

X. Shen, M. Falzon / Regulatory Peptides 113 (2003) 17–29 osteoblast-like cells: a single receptor stimulates intracellular accumulation of both cAMP and inositol triphosphates and increases intracellular free calcium. Proc Natl Acad Sci U S A 1992;89:2732 – 6. Burtis WJ. Parathyroid hormone-related protein: structure, function and measurement. Clin Chem 1992;38:2171 – 83. Wysolmerski JJ, Stewart AF. The physiology of parathyroid hormonerelated protein: an emerging role as a developmental factor. Annu Rev Physiol 1998;60:431 – 60. Orloff JJ, Ganz MB, Nathanson MH, Moyer MS, Kats Y, Mitnick M, et al. A mid-region parathyroid hormone-related protein mobilizes cytosolic calcium and stimulates formation of inositol triphosphate in a squamous cell line. Endocrinology 1996;137:5376 – 85. Fenton AJ, Kemp BE, Hammonds Jr RG, Mitchelhill K, Moseley JM, Martin TJ, et al. A potent inhibitor of osteoclastic bone resorption within a highly conserved pentapeptide region of PTHrP (107-111). Endocrinology 1991;129:3424 – 6. Valin A, Garcia-Ocana A, De Miguel F, Sarasa JL, Esbrit P. Antiproliferative effect of the C-terminal fragments of parathyroid hormonerelated protein, PTHrP (107-111) and (107-139), on osteoblastic osteosarcoma cells. J Cell Physiol 1997;170:209 – 15. Amling M, Neff L, Tanaka S, Inoue D, Kuida K, Wei E, et al. Bcl-2 lies downstream of parathyroid hormone-related peptide in a signaling pathway that regulates chondrocyte maturation during skeletal development. J Cell Biol 1997;136:205 – 13. Kaiser SM, Sebag M, Rhim JS, Kremer R, Goltzman D. Antisensemediated inhibition of parathyroid hormone-related peptide production in a keratinocyte cell line impedes differentiation. Mol Endocrinol 1994;8:139 – 47. Henderson JE, Amikuza N, Warshawsky H, Biasotto D, Lanske BMK, Goltzman D, et al. Nucleolar localization of parathyroid hormone-related peptide enhances survival of chondrocytes under conditions that promote apoptotic cell death. Mol Cell Biol 1995;15: 4064 – 75. Lam MHC, Olsen SL, Rankin WA, Ho PWM, Martin TJ, Gillespie MT, et al. PTHrP and cell division: expression and localization of PTHrP in a keratinocyte cell line (HaCaT) during the cell cycle. J Cell Physiol 1997;173:433 – 46. Dingwall C, Laskey RA. Nuclear targeting sequences—a consensus? Trends Biochem Sci 1991;16:478 – 81. Forti G, Selli C. Prospect for prostatic cancer incidence and treatment by the year 2000. Int J Androl 1996;19:1 – 10. Long RJ, Roberts KP, Wilson MJ, Ercole CJ, Pryor JL. Prostate cancer: a clinical and basic science review. J Androl 1997;18:15 – 20. Feldman D, Skowronski RJ, Peehl D. Vitamin D and prostate cancer. In: Research AICR, editor. Diet and cancer. New York: Plenum; 1995. p. 53 – 63. Chiarodo A. National Cancer Institute roundtable on prostate cancer: future research directions. Cancer Res 1991;51:2498 – 505. Steiner MS. Review of peptide growth factors in benign prostatic hyperplasia and urological malignancy. J Urol 1995;153:1085 – 96. Rabbani SA, Gladu J, Harakidas P, Jamison B, Goltzman D. Overproduction of parathyroid hormone-related peptide results in increased osteolytic skeletal metastasis by prostate cancer cells in vivo. Int J Cancer 1999;80:257 – 64. Deftos LJ. Prostate carcinoma: production of bioactive factors. Cancer 2000;88(Suppl 12):3002 – 8. Goltzman D. Mechanisms of the development of osteoblastic metastases. Cancer 1977;80(Suppl 8):1581 – 7. Iwamura M, Wu R, Abrahamsson PA, di Sant’Agnese PA, Cockett AT, Deftos LJ. Parathyroid hormone-related protein is expressed by prostatic neuroendocrine cells. Urology 1994;43:667 – 74. Kramer S, Reynolds Jr FJ, Castillo M, Valenzuela DM, Thorikay M, Sorvillo JM. Immunological identification and distribution of parathyroid hormone-like protein polypeptides in normal and malignant tissues. Endocrinology 1991;128:1927 – 37. Cramer SD, Peehl DM, Edgar MG, Wong ST, Deftos LJ, Feldman D. Parathyroid hormone-related protein (PTHrP) is an epidermal growth

[34]

[35]

[36]

[37]

[38]

[39]

[40]

[41]

[42]

[43] [44]

[45]

[46]

[47]

[48]

[49]

[50]

[51]

[52]

factor-regulated secretory product of human prostatic epithelial cells. Prostate 1996;29:20 – 9. Wu G, Iwamura M, Di Sant’Agnese A, Deftos LJ, Cockett ATK, Gershagen S. Characterization of the cell-specific expression of parathyroid hormone-related protein in normal and neoplastic prostate tissue. Urology 1998;51:110 – 20. Iwamura M, di Sant’Agnese PA, Wu G, Benning CM, Cockett ATK, Deftos LJ, et al. Immunohistochemical localization of parathyroid hormone-related protein in human prostate cancer. Cancer Res 1993;53:1724 – 6. Iwamura M, Gershagen S, Lapets O, Moynes R, Abrahamsson PA, Cockett AT, et al. Immunohistochemical localization of parathyroid hormone-related protein in prostatic intraepithelial neoplasia. Hum Pathol 1995;26:797 – 801. Asadi F, Farraj M, Sharifi R, Malakouti S, Antar S, Kukreja S. Enhanced expression of parathyroid hormone-related protein in prostate cancer as compared with benign prostatic hyperplasia. Hum Pathol 1996;27:1319 – 23. Tovar Sepulveda VA, Falzon M. Parathyroid hormone-related protein enhances PC-3 prostate cancer cell growth via both autocrine/paracrine and intracrine pathways. Regul Pept 2002;105:109 – 20. Dougherty KM, Blomme EAG, Koh AJ, Henderson JE, Pienta KJ, Rosol TJ, et al. Parathyroid hormone-related protein as a growth regulator of prostate carcinoma. Cancer Res 1999;59:6015 – 22. Iwamura M, Abrahamsson PA, Foss KA, Wu G, Cockett AT, Deftos LJ. Parathyroid hormone-related protein: a potential autocrine growth regulator in human prostate cancer cell lines. Urology 1994;43:675 – 9. Shen X, Falzon M. PTH-related protein modulates PC-3 prostate cancer cell adhesion and integrin subunit profile. Mol Cell Endocrinol 2003;43:675 [in press]. Ivaska J, Heino J. Adhesion receptors and cell invasion: mechanisms of integrin-guided degradation of extracellular matrix. Cell Mol Life Sci 2000;57:16 – 24. Juliano RL, Varner JA. Adhesion molecules in cancer: the role of integrins. Curr Opin Cell Biol 1993;5:812 – 8. Lundstrom A, Holmbom J, Lindqvist C, Nordstrom T. The role of a2h1 and a3h1 integrin receptors in the initial anchoring of MDAMB-231 human breast cancer cells to cortical bone matrix. Biochem Biophys Res Commun 1998;250:735 – 40. Fishman DA, Kearns A, Chilukuri K, Bafetti LM, O’Toole EA, Georgacopoulos J, et al. Metastatic dissemination of human ovarian carcinoma is promoted by a2h1 integrin-mediated interaction with type 1 collagen. Invasion Metastasis 1998;18:15 – 26. Lang SH, Clarke NW, George NJ, Testa NG. Primary prostatic epithelial cell binding to human bone marrow stroma and the role of a2h1 integrin. Clin Exp Metastasis 1977;15:218 – 27. Sato M, Narita T, Kawakami-Kimura N, Higashiyama S, Taniguchi N, Akiyama S, et al. Increased expression of integrins by heparin-binding EGF-like growth factor in human esophageal cancer cells. Cancer Lett 1996;102:183 – 91. Bonaccorsi L, Carloni V, Muratori M, Salvadori A, Giannini A, Carini M, et al. Androgen receptor expression in prostate carcinoma cells suppresses a6h4 integrin-mediated invasive phenotype. Endocrinology 2000;141:3172 – 82. Falzon M, Zong J. The noncalcemic vitamin D analogs EB1089 and 22-oxacalcitriol suppress serum-induced parathyroid hormone-related peptide gene expression in a lung cancer cell line. Endocrinology 1998;139:1046 – 53. Yasuda T, Banville D, Hendy GN, Goltzman D. Characterization of the human parathyroid hormone-like peptide gene. J Biol Chem 1989;264:7720 – 5. Coen DM. The polymerase chain reaction. In: Ausbel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K, editors. Current protocols in molecular biology. New York: Wiley; 1995. Unit 1553. Fraser WD, Robinson J, Lawton R, Durham B, Gallacher SJ, Boyle IT, et al. Clinical and laboratory studies of a new immunoradiometric

X. Shen, M. Falzon / Regulatory Peptides 113 (2003) 17–29

[53]

[54]

[55]

[56]

[57]

[58]

[59]

[60]

[61]

assay for parathyroid hormone-related protein. Clin Chem 1993;39: 414 – 9. Massfelder T, Dann P, Wu TL, Vasavada R, Helwig J-J, Stewart AF. Opposing mitogenic and anti-mitogenic actions of parathyroid hormone-related protein in vascular smooth muscle cells: a critical role for nuclear targeting. Proc Natl Acad Sci U S A 1997;94:13630 – 5. Kaiser SM, Laneuville P, Bernier SM, Rhim JS, Kremer R, Goltzman D. Enhanced growth of a human keratinocyte cell line induced by antisense RNA for parathyroid hormone-related peptide. J Biol Chem 1992;267:13623 – 8. Gujral A, Burton DW, Terkeltaub R, Deftos LJ. Parathyroid hormonerelated protein induces interleukin 8 production by prostate cancer cells via a novel intracrine mechanism not mediated by its classical nuclear localization sequence. Cancer Res 2001;267:2282 – 8. Aarts MM, Rix A, Bringhurst R, Henderson JE. The nuclear targeting signal (NTS) of parathyroid hormone related protein mediates endocytosis and nucleolar translocation. J Bone Miner Res 1999;14: 1493 – 503. Lam MHC, Briggs LJ, Hu W, Martin TJ, Gillespie MT, Jans DA. Importin h recognizes parathyroid hormone-related protein with high affinity and mediates its nuclear import in the absence of importin a. J Biol Chem 1999;274:7391 – 8. Ye Y, Wang C, Du P, Falzon M, Seitz PK, Cooper CW. Overexpression of parathyroid hormone-related protein enhances apoptosis in the rat intestinal cell line, IEC-6. Endocrinology 2001;142:1906 – 14. Ye Y, Falzon M, Seitz PK, Cooper CW. Overexpression of parathyroid hormone-related protein promotes cell growth in the rat intestinal cell line IEC-6. Regul Pept 2001;99:169 – 74. Tovar Sepulveda VA, Shen X, Falzon M. Intracrine PTHrP protects against serum starvation-induced apoptosis and regulates the cell cycle in MCF-7 breasts cancer cells. 2002;143:596 – 606. Nguyen MTA, Karaplis AC. The nucleus: a target site for parathyroid

[62]

[63]

[64]

[65]

[66]

[67]

[68]

[69] [70] [71]

29

hormone-related peptide (PTHrP) action. J Cell Biochem 1998;70: 193 – 9. Lee JW, Juliano RL. a5h1 Integrin protects intestinal epithelial cells from apoptosis through a phosphatidylinositol 3-kinase and protein kinase B dependent pathway. Mol Cell Biol 2000;11:1973 – 87. Mercurio AM, Rabinovitz I. Towards a mechanistic understanding of tumor invasion—lessons from a6h4 integrin. Semin Cancer Biol 2001;11:129 – 41. Rabinowitz I, Nagle RB, Cress AE. Integrin a6 expression in human prostate carcinoma cells is associated with a migratory and invasive phenotype in vitro and in vivo. Clin Exp Metastasis 1995;13:481 – 91. Shaw LM, Chao C, Wewer UM, Mercurio AM. Function of the integrin a6h1 in metastatic breast carcinoma cells assessed by expression of a dominant-negative receptor. Cancer Res 1996;56:959 – 63. Ramos DM, Cheng Y-F, Kramer RH. Role of laminin-binding integrin in the invasion of basement membrane matrices by fibrosarcoma cells. Invasion Metastasis 1991;11:125 – 38. Dedhar S, Saulnier R. Alterations in integrin receptor expression on chemically transformed human cells: specific enhancement of laminin and collagen receptor complexes. J Cell Biol 1990;110:481 – 9. Hemler ME, Crouse C, Sonnenberg A. Association of the VLA alpha 6 subunit with a novel protein. A possible alternative to the common VLA beta 1 subunit on certain cell lines. J Biol Chem 1989;264: 6529 – 35. Guise TA. Parathyroid hormone-related protein and bone metastases. Cancer 1997;80:1572 – 80 [Suppl]. Yoneda T. Cellular and molecular basis of preferential metastasis of breast cancer cells to bone. J Orthop Sci 2000;5:75 – 81. Kostenuik PJ, Singh G, Orr FW. Transforming growth factor h upregulates the integrin-mediated adhesion of human prostatic carcinoma cells to type I collagen. Clin Exp Metastasis 1997;15:41 – 52.