Androgen therapy does not prevent bone loss and arterial calcifications in male rats with chronic kidney disease.

Patients suffering from chronic kidney disease (CKD) often experience bone loss and arterial calcifications. It is unclear if hypogonadism contributes to the development of these complications and whether androgen therapy might prevent them. Male adult rats were randomized into four groups. The first group received standard chow (control), while three other groups were fed a 0.25% adenine/low vitamin K diet (CKD). Two CKD groups were treated with testosterone or dihydrotestosterone (DHT), whereas the control group and one CKD group received vehicle (VEH). CKD animals had 10-fold higher serum creatinine and more than 15-fold higher parathyroid hormone levels compared to controls. Serum testosterone levels were more than two-fold lower in the CKDVEH group compared to control + VEH and CKD + testosterone groups. Seminal vesicle weight was reduced by 50% in CKDVEH animals and restored by testosterone and DHT. CKD animals showed a low bone mass phenotype with decreased trabecular bone volume fraction and increased cortical porosity, which was not rescued by androgen treatment. Aortic calcification was much more prominent in CKD animals and not unequivocally prevented by androgens. Messenger RNA expression of the androgen receptor-responsive genes Acta1 and Col1a1 was reduced by CKD and stimulated by androgen treatment in levator ani muscle but not in the bone or aortic tissue. We conclude that adenine-induced CKD results in the development of hypogonadism in male rats. Androgen therapy is effective in restoring serum testosterone levels and androgen-sensitive organ weights but does not prevent bone loss or arterial calcifications, at least not in the presence of severe hyperparathyroidism.

Stimulation of tumor-associated fatty acid synthase expression by growth factor activation of the sterol regulatory element-binding protein pathway.

Increased expression of fatty acid synthase (FAS) is observed in a clinically aggressive subset of various common cancers and interference with FAS offers promising opportunities for selective chemotherapeutic intervention. The mechanisms by which FAS expression is (up)-regulated in these tumors remain, however, largely unknown. Recently we demonstrated that in LNCaP prostate cancer cells FAS expression is markedly elevated by androgens via an indirect pathway involving sterol regulatory element-binding proteins (SREBPs). Here, we also show that growth factors such as EGF are able to stimulate FAS mRNA, protein and activity. Several observations also indicate that the effects of EGF on FAS expression are ultimately mediated by SREBPs. EGF stimulates SREBP-1c mRNA expression and induces an increase in mature nuclear SREBP-1. Moreover, in transient transfection studies EGF stimulates the transcriptional activity of a 178 bp FAS promoter fragment harboring a complex SREBP-binding site. Deletion or mutation of this binding site abolishes these effects and ectopic expression of dominant negative SREBP-1 inhibits FAS expression and induction in intact LNCaP cells. Given the frequent dysregulation of growth factor signaling in cancer and the key role of SREBP-1 in lipid homeostasis, growth factor-induced activation of the SREBP pathway is proposed as one of the mechanisms responsible for up-regulation of lipogenic gene expression in a subset of cancer cells.

Characterization of newly established testicular peritubular and prostatic stromal cell lines: potential use in the study of mesenchymal-epithelial interactions.

Testicular peritubular and prostatic stromal cells produce extracellular matrix elements and paracrine factors that modulate the cytodifferentiation and function of the corresponding epithelial cells. The present paper describes the establishment and characterization of five rat testicular cell lines with peritubular characteristics and one prostatic stromal cell line. Four peritubular cell lines were isolated after transfection of a mixed peritubular-Sertoli cell culture with a v-myc-containing plasmid. The same immortalization procedure applied to prostatic stromal cells yielded one cell line. An additional testicular cell line arose by spontaneous immortalization during serial subculture. Except for one testicular cell line (RTC-8T1), the morphology of all of the immortalized cell lines strongly resembled that of primary cultures of peritubular and stromal cells. Flow cytometric analysis demonstrated that all cell lines scored positive for alpha-smooth muscle isoactin and negative for cytokeratins, confirming their myofibroblast-like nature. None of the cell lines, however, stained positive for alkaline phosphatase, and androgen receptor expression was also lost. Typical Leydig cell characteristics, such as steroidogenesis, and Sertoli cell markers, such as transferrin secretion, were absent. Coculture of the cell lines with Sertoli cells resulted in the formation of tubular structures. A cell attachment assay and an enzyme-linked immunosorbent assay for fibronectin confirmed the production of extracellular matrix elements by all of the established cell lines. Media conditioned by the cell lines stimulated Sertoli cell transferrin production. The active principle was partially purified and resembled the P-MOD-S-like factors produced by primary cultures of peritubular and stromal cells. It is concluded that the immortalized cell lines have retained several of the characteristics of primary cultures of peritubular and stromal cells and may be useful for further studies on mesenchymal-epithelial interactions in testis and prostate.

Different cells and cell lines produce factors that modulate Sertoli cell function.

Peritubular myoid cells derived from immature rat testes produce factors that modulate Sertoli cell function (P-Mod-S). The secretion of these factors is controlled in part by androgens. Cultured prostatic stromal cells strongly resemble peritubular myoid cells and produce mediators with similar activity. Here we investigated whether myoid cell lines can be used as a source of P-Mod-S-like factors. Rat kidney fibroblast (NRK) and mouse fibroblast (3T3) cell lines were used as non-myoid controls. Surprisingly, serum-free media conditioned by all cell lines studied modulated Sertoli cell function in a similar fashion as media conditioned by peritubular cells (PTCM) or stromal cells (STCM). Using Sertoli cell transferrin secretion as an endpoint for P-Mod-S-like activity, the nature of the active principles involved was further explored. The observed activity could not be explained by residual contamination with fetal calf serum. Moreover, the effects of the conditioned media could not be mimicked by classical growth factors (IGF-I, bFGF, EGF, TGF-beta, NGF, PDGF-BB) added singly or in combination with submaximally effective concentrations of PTCM. Finally, the possibility that conditioned media might indirectly enhance Sertoli cell function by promoting the production or deposition of extracellular matrix elements was made unlikely by the demonstration that the observed effects were not mimicked by Matrigel and were unaffected when Sertoli cells were seeded on Matrigel. Superdex 75 chromatography after analytical reversed-phase chromatography indicates that the factors from different origin have a similar size (45-50 kDa). It is concluded that mediators with P-Mod-S-like activity are produced by various cells and cell lines both with and without smooth muscle cell characteristics. Whether the active principles involved are really identical requires further investigation.

The role of cell-cell interactions in androgen action.

Androgen-regulated mesenchymal-epithelial interactions play an important role during embryonic development of the male urogenital tractus. Studies on the effects of androgens on cultured testicular cells derived from the immature rat testis indicate that, even during postnatal life, similar interactions may be instrumental for normal androgen action. Androgen receptors are found in epithelial Sertoli cells as well as in mesenchymal peritubular cells. The effects of androgens on isolated Sertoli cells, however, are limited. Coculture with peritubular cells increases the sensitivity and/or the responsiveness of a number of Sertoli cell parameters (transferrin, ABP, aromatase activity) to androgens. This effect is at least in part mediated by the secretion of one or more diffusible factors (P-Mod-S) by the peritubular cells. We investigated whether such indirect effects of androgens, relying on mesenchymal-epithelial interactions are also observed in other androgen target tissues. To this end stromal cells were isolated and cultured from the immature rat ventral prostate and the production of factors with P-Mod-S activity was monitored using Sertoli cells as the test system. Under coculture conditions these stromal cells stimulate Sertoli cell transferrin secretion in an androgen-regulated fashion, exactly as peritubular cells. This stimulatory effect is related in part to the collaborative (and androgen-independent) deposition of an extracellular matrix and in part to the secretion of an androgen-regulated diffusible mediator. This mediator has the same physicochemical characteristics as P-Mod-S and it affects other Sertoli cell parameters (ABP, aromatase activity, inhibin, cGMP) in the same way as P-Mod-S. Cultured stromal and peritubular cells look very similar and stain positive after immunostaining for alpha-smooth muscle isoactin. Tissue sections suggest that these cells may be derived from myoid peritubular cells in the testis and similar periacinar cells in the prostate. The hypothesis is advanced that P-Mod-S may be a more universal mediator of indirect effects of androgens in diverse target tissues and that this factor is derived from myoid cells closely associated with the epithelial component.

Morphological and functional similarities between cultured prostatic stromal cells and testicular peritubular myoid cells.

A number of androgen effects on epithelial cells may be mediated by androgen-regulated paracrine factors produced by underlying mesenchymal cells. In previous studies we demonstrated that prostatic stromal cells and testicular peritubular cells, derived from immature rats, produce mediators of androgen action with identical effects on Sertoli cells. In the present paper we further compared the morphological and functional characteristics of both mesenchymal cell types. Cultured prostatic stromal cells and testicular peritubular cells look identical under phase-contrast microscopy, share the ability to form tubular structures and "balls" when cocultured with Sertoli cells, and contain proteins immunoreactive with an antiserum against alpha-smooth muscle isoactin. Two-dimensional gel electrophoresis shows that the pattern of proteins produced by both cell types is nearly identical. Conditioned media from stromal and peritubular cells contain a factor that stimulates transferrin and cGMP production in Sertoli cells. The behavior of the active principle in the media from both cell types is comparable. On reverse-phase HPLC the elution profile of this factor is comparable for media from both cell types. In conclusion, these data point to a striking similarity in the morphological and functional characteristics of mesenchymal cells cultured from the prostate and testis.

Effects and characterization of paracrine factors produced by human prostate stromal cells in bioassays using rat Sertoli cells, LNCaP tumor cells, and cultured prostate epithelial cells.

BACKGROUND: Prostatic stroma affects both proliferation and differentiation of epithelial cells but the factors involved remain poorly understood. In order to identify and characterize potential paracrine mediators, we studied the effects of human prostate fibroblast-conditioned media (PFCM) in three bioassay systems. METHODS: The first bioassay uses transferrin secretion by cultured rat Sertoli cells as an endpoint for differentiating activity. Factors active in this (heterologous) assay were compared to PModS, a mediator of mesenchymal-epithelial interactions in the testis, also produced by rat prostate stromal cells. The two other (homologous) bioassays use LNCaP tumor cells or subcultured human prostate epithelial cells (PEC) as targets. Differentiation is evaluated by prostate-specific antigen (PSA) secretion and reverse transcriptase-polymerase chain reaction (RT-PCR) for a number of markers of epithelial function. Proliferation is assayed by measurements of DNA and thymidine incorporation. RESULTS: PFCM markedly stimulates transferrin production by Sertoli cells. The main factor(s) involved are acid stable and bind to heparin. However, both their size (approximately 37 kDa) and their behavior on reversed-phase chromatography differ from that of PModS. Although PFCM increases total RNA content of LNCaP, it does not increase or restore differentiated function of LNCaP or PEC. Proliferative effects are observed in LNCaP and these effects cannot be neutralized by an antiserum directed against basic fibroblast growth factor (bFGF). Antiproliferative effects are observed in PEC and these effects are largely due to transforming growth factor-beta (TGF-beta). CONCLUSIONS: PFCM provokes differentiating effects in a Sertoli cell bioassay, but unlike with rat stromal cells, the factor(s) involved differ from PModS. In the two homologous systems studied, differentiating effects could not be demonstrated and discordant effects were noted on proliferation. Various bioassay systems will be required to identify the spectrum of mediators present in PFCM.

Effect of androgens on the germ cell-depleted testes of prenatally irradiated rats.

To study the effect of androgens on somatic testicular cells, rats were rendered germ cell depleted by prenatal irradiation (RX). Adult RX rats were treated with a desensitizing dose of a GnRH agonist (GnRHa; Zoladex), combined with an antiandrogen (Nilutamide) to preclude all androgen effects, or combined with testosterone or hCG to restore androgen action. The effect of these treatments for 3 weeks on the weight of testes and accessory sex glands, hormones (LH, FSH, testosterone, inhibin), testicular proteins, the pattern of incorporation of [35S]-methionine into testicular proteins (studied by two dimensional gel electrophoresis) and steady state mRNA levels for transferrin and androgen-binding protein (ABP) were evaluated. Combined treatment with GnRHa and antiandrogen virtually eliminated gonadotrophins, androgens and androgen effects. Testicular weight was reduced to 50% of that observed in RX controls. Treatment with GnRHa and testosterone resulted in supraphysiological levels of testosterone and testicular weights comparable to those observed in RX controls. FSH levels in these animals, however, were in the normal range. A low dose of hCG also restored testicular weight in the presence of low concentrations of serum testosterone and low normal levels of FSH. Neither polyacrylamide gel electrophoresis of total testicular proteins nor two dimensional gel electrophoresis of [35S]-methionine labelled proteins revealed striking changes in distinct testicular proteins as a result of androgen withdrawal or androgen treatment. Dot blot hybridization showed a three-fold increase in the mRNA level for ABP (expressed per microgram total RNA) in the Sertoli cell enriched testes of RX rats. This level was barely influenced by androgen withdrawal or androgen administration. The mRNA level for transferrin was increased six-fold in RX rats. A 50% reduction of this level was observed after combined treatment with GnRHa and antiandrogen. It is concluded that, in the germ cell-depleted testis, the major effect of androgens is an overall increase in protein and RNA synthesis rather than a very important and selective increase of a few gene products.