Unique domain functions of p63 isotypes that differentially regulate distinct aspects of epidermal homeostasis.

p63 is critical for squamous development and exists as multiple isotypes of two subclasses, TA and DeltaN. DeltaNp63 isotypes can antagonize transcription by TAp63 and p53, and are highly expressed in squamous cell cancers. Using mouse keratinocytes as a biological model of squamous epithelium, we show that multiple p63 isotypes, DeltaN- and TA-containing, are expressed and differentially modulated during in vitro murine keratinocyte differentiation. DeltaNp63alpha declines with Ca2+-induced differentiation, while a smaller DeltaN-form, DeltaNp63s, persists, suggesting unique functions of the two DeltaN-forms. To investigate the impact of dysregulated p63 expression that is observed in cancers and to define the biological contribution of the different domains of the p63 isotypes, DeltaNp63alpha, DeltaNp63p40, TAp63alpha, TAp63gamma or beta-galactosidase were overexpressed in primary murine keratinocytes. Microarray, RT-PCR and western blot analyses revealed that overexpression of DeltaNp63p40, which lacks the entire alpha-tail present in DeltaNp63alpha, permits expression of a full panel of differentiation markers. This is in contrast to overexpression of the full-length DeltaNp63alpha, which blocks induction of keratin 10, loricrin and filaggrin. These findings support a role for the alpha-tail of DeltaNp63alpha in blocking differentiation-specific gene expression. Overexpression of either TAp63 isotype permits keratin 10 and loricrin expression, thus the alpha-terminus requires the cooperation of the DeltaN domain in blocking early differentiation. However, both TA isotypes block filaggrin induction. The DeltaN-terminus is sufficient to maintain keratinocytes in a proliferative state, as both DeltaN forms block Ca2+-mediated p21WAF1 induction and S-phase arrest, while sustaining elevated PCNA levels. No alteration in cell cycle regulation was observed in keratinocytes overexpressing TAp63alpha or TAp63gamma. Clarifying the functional distinctions between p63 isotypes and domains will help to elucidate how their dysregulation impacts tumor biology and may suggest novel therapeutic strategies for modulating behavior of tumor cells with altered expression of p53 family members.

The WFDC1 gene encoding ps20 localizes to 16q24, a region of LOH in multiple cancers.

We previously identified ps20 protein as a secreted growth inhibitor and purified the protein from fetal rat prostate urogenital sinus mesenchymal cell conditioned medium. The rat cDNA was subsequently cloned, and ps20 was found to contain a WAP-type four-disulfide core motif, indicating it may function as a protease inhibitor. We now report cloning and characterization of the mouse ps20 gene (designated Wfdc1), the human homolog cDNA, and the human gene (designated WFDC1). Both the mouse and human WFDC1 genes consist of seven exons and encode respective ps20 proteins sharing 79.1% identity and nearly identical WAP motifs in exon 2. The WFDC1 gene was mapped by FISH analysis to human Chromosome (Chr) 16q24, an area of frequent loss of heterozygosity (LOH) previously identified in multiple cancers including prostate, breast, hepatocellular, and Wilms' tumor. Identification and characterization of the WFDC1 gene may aid in better understanding the potential role of this gene and ps20 in prostate biology and carcinogenesis.

Transforming growth factor-beta1 induces nuclear to cytoplasmic distribution of androgen receptor and inhibits androgen response in prostate smooth muscle cells.

Stromal-epithelial interactions in the prostate gland are dependent on androgen regulation of prostate stromal cells, yet little is known about androgen action in these cell types. Recent reports have demonstrated that androgen-regulated gene transcription can be stimulated or inhibited by certain growth factors, indicating cross-talk mechanisms. To address potential cross-talk in signaling pathways between androgen and transforming growth factor-beta1 (TGFbeta1) in prostate stromal cells, the PS-1 prostate smooth muscle cell line was examined. In the presence of physiological concentrations of androgen, PS-1 cell proliferation was stimulated, and androgen receptor (AR) exhibited a nuclear localization pattern. The addition of TGFbeta1 (25 pM) was capable of blocking androgen-induced proliferation, but had no direct effect in cultures without androgen. Immunocytochemistry to localize AR subcellular distribution showed that TGFbeta1 (5-100 pM) altered the distribution of AR from the nucleus to the cytoplasm. Other growth factors, including fibroblast growth factor-2, epidermal growth factor, and TGFbeta2 had no effect on AR distribution. The TGFbeta1-induced nuclear to cytoplasmic change in receptor localization was rapid (initiated within 30 min), was neutralized by TGFbeta1 antibodies, did not require new protein synthesis, and was complete by 6 h. Removal of TGFbeta1 from the culture medium resulted in a rapid redistribution of AR to the nucleus, indicating reversible mechanisms. Northern analysis of the ddp17 marker transcript for androgen action in PS-1 cells showed that androgen-stimulated ddp17 expression was inhibited in the presence of TGFbeta1 (25 pM). TGFbeta1 induced a similar nuclear to cytoplasmic distribution of AR in primary cultures of rat prostate stromal cells. TGFbeta1, however, had no effect on AR distribution in either the LNCaP prostatic carcinoma cell line or the DDT1MF-2 leiomyosarcoma cell line. Specific cross-talk between TGFbeta1 and AR signaling pathways in prostate stromal cells may play a significant role in prostate development and stromal cell response in carcinoma progression.

Localization of transforming growth factor-beta1 and type II receptor in developing normal human prostate and carcinoma tissues.

Transforming growth factor-beta1 (TGF-beta1) is implicated in prostate development, and elevated expression of TGF-beta1 has been correlated with prostate carcinogenesis. In this study, cell type specificity of TGF-beta1 and TGF-beta receptor Type II (RcII) protein expression was determined by immunocytochemistry in human normal prostate and compared to prostate carcinoma tissues. Heterogeneous localization patterns of LAP-TGF-beta1 (TGF-beta1 precursor) and RcII were observed in both epithelial and mesenchymal cells in fetal prostate, with LAP-TGF-beta1 localizing to more basal epithelial cells. Homogeneity of LAP-TGF-beta1 staining was increased in neonatal, prepubertal, and adult prostate, with elevated immunoreactivity noted in epithelial acini relative to stromal tissue for both LAP-TGF-beta1 and RcII proteins. In stromal tissues, RcII cell localization exhibited staining patterns nearly identical to smooth muscle alpha-actin. In prostate carcinoma, LAP-TGF-beta1 localized to carcinoma cells with an increased staining heterogeneity relative to normal prostate. In contrast to normal epithelial cells, carcinoma epithelial cells exhibited low to nondetectable RcII staining. Stromal cell staining patterns for LAP-TGF-beta1 and RcII in carcinoma, however, were identical to those of normal prostate stromal cells. These studies implicate both epithelial and stromal cells as sites of TGF-beta1 synthesis and RcII localization in the developing and adult normal human prostate. In addition, these data indicate a loss of epithelial expression of RcII concurrent with altered LAP-TGF-beta1 expression in human prostate carcinoma cells.

Molecular cloning and expression of ps20 growth inhibitor. A novel WAP-type "four-disulfide core" domain protein expressed in smooth muscle.

We previously reported the purification of ps20 (Rowley, D. R., Dang, T. D., Larsen, M., Gerdes, M. J., McBride, L., and Lu, B. (1995) J. Biol. Chem. 270, 22058-22065), a urogenital sinus mesenchymal cell secreted protein having growth-inhibitory properties. We report here cloning of the 1.03-kilobase rat ps20 cDNA clone from the PS-1 (adult rat prostate smooth muscle) cDNA library. Partial clones were obtained by nested polymerase chain reaction with degenerate primers, and full-length ps20 cDNA clones were isolated by plaque hybridization. Sequence analysis revealed that ps20 protein contains a WAP-type "four-disulfide core" motif and is a novel member of the WAP signature protein family composed primarily of secreted serine protease inhibitors. Native ps20 immunoprecipitated from smooth muscle cells and recombinant ps20 both resolved on SDS-polyacrylamide gel electrophoresis with apparent molecular mass of 27-29 kDa under reducing conditions and 21-23 kDa under non-reducing conditions, respectively. Stable ps20-transfectant COS-7 cell lines secreted ps20 and were growth-inhibited relative to mock transfectants. In addition, COS-7 and prostate carcinoma PC-3 cells were growth-inhibited by bacterially expressed ps20. Northern analysis indicated differential expression by tissue with highest expression in the heart. Immunohistochemical localization of ps20 protein showed cell-specific expression by both visceral and vascular smooth muscle in all tissues, including the prostate gland. These results indicate ps20 is a novel growth-regulatory member of the WAP signature family expressed by smooth muscle cells.

Androgen-regulated proliferation and gene transcription in a prostate smooth muscle cell line (PS-1).

Androgens play a key role in directing stromal-epithelial interactions in prostate gland development, in maintenance of adult phenotype, and in disease progression. To address the molecular mechanisms of androgen action in prostate stromal cells and the genes regulated by androgens in this cell type, a stromal cell line (PS-1) was developed from rat ventral prostate. The PS-1 cell line was adapted to chemically defined media and characterized as smooth muscle based on expression of desmin, smooth muscle alpha-actin, and nuclear androgen receptor, markers for prostate smooth muscle. To examine steroid hormone regulation, PS-1 cells were analyzed for response to a panel of steroid hormones in serum-free, chemically defined media. PS-1 cells were significantly growth stimulated by physiological concentrations of androgens (10nM) relative to other steroids tested. To ascertain whether this cell line could be used to examine androgen-regulated gene expression, differential display PCR was used to demonstrate the presence of androgen-regulated transcripts and provide the initial steps in cloning these genes. Replicate differential display PCR analyses showed consistent androgen stimulation of 16 messenger RNA species. Northern analysis confirmed androgen regulation of 6 species. Sequence analysis of each indicated no regions of significant homology or direct matches to existing sequences. Further study of the PS-1 stromal cell line and androgen-regulated genes identified here will provide a novel in vitro system for defining molecular mechanisms of androgen action in prostate stromal cells and the significance of stromal androgen-regulated genes to stromal-epithelial interactions.

Purification of a novel protein (ps20) from urogenital sinus mesenchymal cells with growth inhibitory properties in vitro.

Our previous studies have characterized mesenchyme-derived proteins to identify biologically active proteins and novel markers for stromal cell paracrine action relative to stromal-epithelial interactions. Previous reports have characterized properties of a growth inhibitory activity (to bladder and prostatic epithelial cells), secreted by U4F fetal rat urogenital sinus mesenchymal cells, not cross-reactive with antibodies to known cytokines, and provisionally termed UGIF. The present study reports the characterization, purification, and biological properties of a 20-21-kDa protein responsible for UGIF activity. The 20-21-kDa protein (termed ps20) was purified to near homogeneity, the amino-terminal sequence was determined, and biological properties were characterized in vitro. Amino-terminal sequence analysis indicated no direct matches or regions of homology with known proteins. Purified ps20 induced a linear and saturable inhibition of [3H]thymidine incorporation in PC-3 prostatic carcinoma cells (half-maximal activity at 2.6 nM), inhibited cell proliferation (increased population doubling time from 19.8 to 25.8 h), and induced a 210% stimulation in the synthesis of secreted proteins. These data suggest that ps20 may be a candidate paracrine effector protein and may play a role in stromal-epithelial cell interaction in the prostate gland.

Beta-2 microglobulin is mitogenic to PC-3 prostatic carcinoma cells and antagonistic to transforming growth factor beta 1 action.

Previous studies have identified a M(r) 12,000 protein in rat prostatic stromal cell-conditioned medium with growth stimulatory activity to human prostatic carcinoma cells as a direct match with beta 2-microglobulin (beta 2-m). The present study was conducted to characterize the activities of human beta 2-m directly, using commercially available, purified human beta 2-m. Beta 2-m was assayed for growth stimulatory activity to human PC-3 prostatic carcinoma cells and rat PS-1 prostatic stromal cells and for antagonistic activity to transforming growth factor beta 1 (TGF-beta 1)-induced growth inhibitory actions. Beta 2-m acted to stimulate [3H]thymidine incorporation in PC-3 cells in a linear, concentration-dependent and saturable manner in serum-free medium. Beta 2-m stimulated cell proliferation and significantly decreased population doubling times in both PC-3 and PS-1 cell lines. At half-maximal concentrations of TGF-beta 1 and lower, beta 2-m acted in a concentration-dependent, antagonistic manner, acting to stimulate growth-inhibited PC-3 cells to fully neutralize TGF-B1 activity. In contrast, cells exposed to maximum activity TGF-beta 1 concentrations were refractory to beta 2-m action, regardless of the concentration tested. This represents the first report to demonstrate a growth-stimulatory activity of B2-m with carcinoma/epithelial cells and to show beta 2-m antagonistic activity to TGF-B1 growth-induced inhibition. Beta 2-m has been shown previously to associate with hormone/growth factor receptors. Together, these data suggest that beta 2-m may play a role in modulating cell proliferation, possibly through modification of ligand/receptor kinetics. Owing to the elevation of both beta 2-m and TGF-beta 1 in many dysplastic-neoplastic conditions, beta 2-m may be relevant to mechanisms of abnormal proliferation disorders and in modulating TGF-beta 1 mechanisms of actions.