Leland W. K. Chung

Hormone-induced morphogenesis and growth: role of mesenchymal-epithelial interactions.

Publisher Summary nThis chapter explains the role of mesenchymal–epithelial interactions in hormone induced morphogenesis and growth. The mechanism of steroid hormone action is thought to involve specific high-affinity receptor proteins. The hormone enters the cell, binds to the cytoplasmic receptor, which after activation translocates to the nucleus. The hormone–receptor complex in turn binds to nuclear acceptor sites on the chromatin. This activates a variety of metabolic processes, the most important being the stimulation of messenger RNA (mRNA) synthesis and the ultimate production of new proteins. The first indication that androgens can elicit their effects upon epithelial morphogenesis via the mediation of mesenchymal cells comes from studies in which urogenital epithelia from the embryonic seminal vesicle or urogenital sinus are grown in association with either urogenital mesenchyme or with non-target integumental mesenchyme. Urogenital mesenchyme induces specific epithelial morphogenesis, growth, and function within the genital tract and that the hormonal sensitivity of these morphogenetic processes resides in the mesenchyme that invariably contains nuclear hormone receptors. As morphogenetic processes are cyclic in adult genital tracts of many species, developmental properties are expressed in adulthood and, for this reason, appear to play a regulatory role in abnormal epithelial differentiation including carcinogenesis.

Stromal-epithelial interactions--I. Induction of prostatic phenotype in urothelium of testicular feminized (Tfm/y) mice.

Heterotypic tissue recombinants (UGM+/+ + BLETfm) were prepared with embryonic wild-type urogenital sinus mesenchyme (UGM+/+) and epithelium of the urinary bladder (BLETfm) of adult Tfm/Y mice. Following 1 month of growth in male hosts, the recombinants contained prostatic acini containing secretory product. In UGM+/++ BLETfm recombinants, secretory activity, epithelial cytodifferentiation, and DNA synthesis were stimulated by testosterone propionate and antagonized with cyproterone acetate. Total cellular protein synthesis was quantitatively and qualitatively similar to that of the prostate and distinct from that of bladder. These data suggest that Tfm epithelium is capable of expressing a prostatic, androgen-sensitive differentiation when grown in association with wild-type urogenital sinus mesenchyme.

Androgen-induced biochemical responses in epithelium lacking androgen receptors: Characterization of androgen receptors in the mesenchymal derivative of urogenital sinus

Heterotypic tissue recombinants, composed of adult bladder epithelium obtained from testicular feminization syndrome mice (TfmBLE) and embryonic wild-type rat urogenital sinus mesenchyme (UGM) were grown as subcapsular renal grafts in male athymic nude mice for 30 days. The resultant prostatic tissue that developed was subjected to extensive biochemical analyses for androgen receptors. From previous autoradiographic studies it was shown that UGM possesses androgen receptors and induces the TfmBLE to form prostatic ductal-acinar epithelium that lacks androgen receptors. The purpose of the present study is to biochemically characterize the mesenchymal androgen receptors. According to results obtained by autoradiographic analyses, androgen receptors are expressed in the mesenchyme of tissue recombinants. Cytosolic androgen receptors analyzed by the Scatchard method utilizing R-1881 as a ligand showed that the dissociation constant (Kd) of androgen receptors localized within the mesenchyme of the tissue recombinants differs from that found in the mouse prostate gland, but resembles that found in the rat ventral prostate (VP). Sucrose density gradient analysis of the cytosol androgen receptors showed the presence of 8 S [3H]dihydrotestosterone (DHT) binding component under the low salt condition. The mesenchymal androgen receptors are capable of translocating efficiently from cytosol to nuclear compartment, seemingly unaffected by adjacent TfmBLE. The quantity of both cytosol and nuclear androgen receptors expressed in tissue recombinants is only about 1/3 of that found in the rat VP and is in agreement with the morphometric analysis of tissue recombinants, which indicated that about 1/3 of the cells in the tissue recombinants are stromal cells. These results extend previous autoradiographic results and further suggest that androgen receptors present in the mesenchymal compartment may be necessary for the expression of androgen-elicited responses in the TfmBLE that lacks androgen receptors. In addition, this study confirms a novel model system for the study of mesenchymal androgen receptors in tissues unmolested by mechanical or enzymatic dissociation.

Tissue Interactions and Prostatic Growth: A New Mouse Model for Prostatic Hyperplasia

The pathologic process of human benign prostatic hyperplasia (BPH) has been suggested by McNeal as a reawakening of embryonic growth potential of the adult stroma. The proliferating stromal element in the periurethral region of human prostate can lead to the ingrowth and formation of new epithelial acini and ducts.* Experimental support of the reactivation of embryonic growth potential hypothesis was provided by Cunha and his collaborators who showed that embryonic urogenital sinus mesenchyme (equivalent to embryonic prostatic stroma) is a potent inductor for the growth and differentiation of adult bladder e~i the l ia .~ .~ Evidence directly supporting that adult prostatic cells indeed responded to growth induction by fetal urogenital sinus was provided by Chung e? al.V6 and Rocco et a1.l They observed extensive overgrowth in chimeric prostates prepared by xenografting fetal urogenital sinus tissues directly into the adult host prostates. These results were confirmed recently by Cunha e? a1.* who demonstrated growth responsiveness of adult mouse dorsolateral prostate to embryonic rat urogenital sinus mesenchyme induction in tissue recombinants. The purpose of the present study is to document that fetal urogenital sinus tissues have growth-promoting activity both in situ and in cultured cells. The respective roles of fetal urogenital sinus mesenchyme (UGM) and its epithelium (UGE) on the overall expression of growth-inductive capability by the intact fetal urogenital sinus (UGS) will be discussed. The homologous chimeric prostate induced in rodent species (mouse and rat) represents a new model for prostatic hyperplasia.j Cellular interactions between fetal and adult prostatic tissues may provide significant insight in elucidating the regulatory mechanisms of adult prostatic growth and differentiation.

Androgen metabolism in tissue recombinants composed of adult urinary bladder epithelium and urogenital sinus mesenchyme.

Epithelium of the adult mouse urinary bladder (BLE) was experimentally combined with mesenchyme of the urogenital sinus (UGM) and grown in intact male hosts to produce prostate-like glandular structures. To determine the extent to which the BLE is altered in a functional sense by inductive influences from UGM, investigations into the in vitro metabolism of tritiated testosterone (T) were undertaken. An isocratic high performance liquid chromatographic (HPLC) method was developed in order to separate the metabolites of T in mouse bladder, prostate and UGM + BLE tissue recombinants. Using a C-18 reversed phase column and a tetrahydrofuran (20): methanol (40): H2O (40) mobile phase, efficient and rapid separation of T, dihydrotestosterone, 3 alpha-androstanediol, androstenedione, androstanedione and androsterone was achieved. The identities of the radiolabeled T metabolites were confirmed by recrystallization to constant specific activity. The results of the present study revealed that tissue recombinants expressed testosterone metabolic profiles only partially toward that of the adult prostate. For example, percentage formation of 5 alpha-androstanedione, 3 alpha-androstanediol and unknown polar metabolites in the UGM + BLE resembled the prostate and differed significantly from the urinary bladder. Conversely, formation of the 3 beta-androstanediol and androsterone from testosterone resembled the urinary bladder and differed from the formation of these metabolites in the prostate. These results suggest that in contrast to histomorphology, androgen-induced DNA synthesis, androgen receptor binding activity and total tissue two-dimensional gel electrophoretic protein profiles, androgen metabolic profiles in the tissue recombinants showed only partial transformation into prostatic phenotypes. Analysis of steroid-metabolic profiles, therefore, may represent an exquisite and sensitive method to assess gene expression in various hormone-responsive target tissues.

Evidence for a Soluble Fetal Urogenital Sinus-derived Growth Factor(s)

The inductive capacity of fetal urogenital sinus mesenchyme (UGM) has been established previously.’** Recently, our laboratory described a new rodent model for prostatic hyperplasia whereby a marked prostatic overgrowth (10to 20-fold) can be induced in situ by direct implantation of either intact fetal urogenital sinus (UGS) or UGM into the adult prostate gland^.^,^ In an effort to elucidate the underlying mechanism for the induction of prostatic overgrowth by fetal urogenital sinus tissues, a tissue culture system was developed whereby the UGS was co-cultured with non-confluent primary culture cells derived from adult rat ventral prostatic stroma (Wistar strain, three to five months). To test for the possible existence of a soluble fetal urogenital sinusderived growth factor(s), attempts were made to prevent cell-cell contact during the culturing procedures. Modified procedures resulted in the proliferation of two cell populations (i.e., adult ventral prostatic stroma and fetal UGS-derived embryonic cells) interacting only through the exchange of certain soluble and diffusible factor( s). In brief, experiments were performed as follows. Ventral prostates were excised aseptically from the adult male rats. Ventral prostatic stromal cells were isolated from minced tissues by mild collagenase digesti~n.~ They were plated in 35 mm plastic dishes at 6.5 x lo-’ cells/dish. Twelve hours later, UGSs removed from 18-day-old Nb rat embryos’ were co-cultured in small glass dishes (one UGS/dish) which had been placed within the larger plastic 35 mm dishes containing the previously seeded adult stroma. Cells were then grown under standard conditions of 95% air, 5% C 0 2 in F12K media containing 20% fetal calf serum, 5 pg/ml transferrin, 5 pg/ml bovine insulin, 0.1 pg/ml dihydrotestosterone, 1 mglml bovine serum albumin, and penicillin (50 units)-streptomycin (50 pg) per ml. Medium was changed every 48 hours and growth was assessed immediately afterwards by histological and biochemical methods as discussed below. FIGURE 1 provides morphological evidence of increased proliferation of adult stroma (comparison with control stroma, FIGURE la) co-cultured with the intact fetal UGS and its derived cells. Areas selected for their representative histomorphology were obtained by staining the culture dishes with Giemsa dye seven days after the control and UGS-induced growth. In addition to increased cell number observed under fetal UGS-induced growth, the induced stromal cells were also larger and appeared more “stretched,” a condition that has been previously observed to indicate a more tenacious, trypsin-resistant attachment to the substrata.6 We have also observed increased size of cell nuclei and number of nucleoli in the UGS co-cultured adult stromal cells. These results suggest polyploidy in the induced cells. FIGURE 1 demonstrates the outgrowth of both embryonic epithelia (c) as well as embryonic mesenchyme (d) from the UGS in culture,

Role of Androgen Receptors in Stromal-Epithelial Interactions in Prostatic Cancer

Since the demonstration in 1941 by Huggins and Hodges on the endocrine dependency of neoplastic human prostate [1], this relationship has formed the foundation for investigations into the mechanism(s) of steroid hormone action on hormone-responsive target tissues. The discovery of the high affinity and limited capacity of estrogen receptors in immature rat uterus by Jensen [2] has firmly established the initial steps of steroid hormone and target tissue interactions. The rigid structural requirements of steroid hormones, their specific binding to cytoplasmic receptor proteins to form receptor protein complexes, and their ability to elicit specific biological responses have been documented at molecular [3, 4], genetic [5, 6], and developmental [7, 8] levels. Similar receptor proteins for androgens [9, 10], estrogens [11, 12], and/or progestins [13] have been demonstrated to exist in the prostate gland of virtually all species studied.