Mullerian-inhibiting substance regulates NF–B signaling in the prostate in vitro and in vivo
Dorry L. Segev*, Yasunori Hoshiya*, Makiko Hoshiya, Trinh T. Tran, Jennifer L. Carey, Antonia E. Stephen, David T. MacLaughlin, Patricia K. Donahoe, and Shyamala Maheswaran†
Pediatric Surgical Research Laboratories, Massachusetts General Hospital, and Harvard Medical School, Boston, MA 02114
Contributed by Patricia K. Donahoe, November 8, 2001 Mullerian-inhibiting substance (MIS) is a member of the transform- the male as the prostatic utricle, around which the prostate forms ing growth factor  superfamily, a class of molecules that regulates later in development (12). Based on these observations, we growth, differentiation, and apoptosis in many cells. MIS type II tested the hypothesis that MIS type II receptor may be expressed receptor in the Mullerian duct is temporally and spatially regulated in the prostate gland and that prostate may be an additional during development and becomes restricted to the most caudal target tissue for the action of MIS. In this article, we demonstrate ends that fuse to form the prostatic utricle. In this article, we have MIS type II receptor expression in the prostate glands of adult demonstrated MIS type II receptor expression in the normal pros- mice and human prostate tissue and cancer cell lines. As with tate, human prostate cancer cell lines, and tissue derived from breast cancer cells, MIS induced NF-B signaling and the patients with prostate adenocarcinomas. MIS induced NF– B DNA immediate early gene IEX-1S in prostate cancer cells and murine binding activity and selectively up-regulated the immediate early prostate glands in vivo. Abrogation of NF-B activity with gene IEX-1S in both androgen-dependent and independent human dominant negative IB-␣ ablated MIS-mediated molecular prostate cancer cells in vitro. Dominant negative IB␣ expression events and inhibition of prostate cancer cell growth, suggesting ablated both MIS-induced increase of IEX-1S mRNA and inhibition that the prostate is a likely target for the action of MIS. of growth, indicating that activation of NF–B signaling was required for these processes. Androgen also induced NF–B DNA Materials and Methods binding activity in prostate cancer cells but without induction of Cell Lines, Reagents, and Growth Inhibition Assays. IEX-1S mRNA, suggesting that MIS-mediated increase in IEX-1S was LNCaP cells independent of androgen-mediated signaling. Administration of were grown in RPMI 1640 containing 10% FBS, glutamine, and penicillin͞streptomycin. DU145 cells were grown in Eagle’s MIS to male mice induced IEX-1S mRNA in the prostate in vivo, ͞ suggesting that MIS may function as an endogenous hormonal minimum essential medium containing Earle’s BSS 2mM ͞ ͞ regulator of NF–B signaling and growth in the prostate gland. L-glutamine 1.0 mM sodium pyruvate 0.1 mM nonessential aminoacids͞1.5 g/liter sodium bicarbonate͞10% FBS and pen- ͞ IEX-1 icillin streptomycin. PC3 cells were grown in Ham’s F12K medium supplemented with 10% FBS͞2mML-glutamine͞1.5 g/liter sodium bicarbonate and penicillin͞streptomycin. To mea- ullerian-inhibiting substance (MIS) belongs to the trans- Mforming growth factor  (TGF-) superfamily, which also sure inhibition of cell proliferation, cultures were grown in the includes activins, inhibins, and the bone morphogenetic proteins. presence or absence of 35 nM MIS for 4 days, and cell numbers The Mullerian duct, the anlagen of the uterus, Fallopian tubes, were determined by using a Coulter counter. To determine the and upper vagina in the female, regresses in the presence of MIS effects of androgen, cells were grown in medium containing 7% in male embryos (1). The MIS type II receptor, a transmembrane charcoal-stripped serum devoid of androgen (Gemini Biological serine threonine kinase, is expressed at high levels in the Products, Calabasas, CA) for 2–5 days and treated with 10 nM ␣ Mullerian duct and in the embryonic and adult gonads (2). 5 -dihydrotestosterone (DHT, Sigma). Lower levels of receptor expression were detected in the mam- Human recombinant MIS (rhMIS) was collected from growth mary gland and embryonic rat lungs (3–5), which show func- media of Chinese hamster ovary cells transfected with the human tional responses to MIS. MIS gene and purified as described (13). The binding of MIS to its receptor initiates a signaling cascade that depends on the recruitment of type I receptors ALK2 and In Situ Hybridization, Electrophoretic Mobility-Shift Assay (EMSA), and ALK6 (6–8). We recently demonstrated that MIS inhibited the Northern Blot. Timed pregnant rats were purchased from Harlan growth of breast cancer cells in vitro through induction of NF–B Breeders (Indianapolis), 16-day embryos were harvested, and in (4), a family of transcription factors that includes p50, p65, p52, situ hybridization to detect MIS type II receptor was performed and c-rel. These transactivators form either homodimers or as described (6). heterodimers that are retained in the cytoplasm by a class of Nuclear proteins were harvested, and EMSA was performed inhibitor proteins, IB-␣,IB-,IB-␥, and IB-. Nuclear as described (4). Total RNA isolated from cells was probed with translocation of NF–B after phosphorylation and degradation radiolabeled IEX-1, glyceraldehyde-3-phosphate dehydroge- of IB results in changes in gene expression (9, 10). MIS nase, or 18S ribosomal RNA as described (4). selectively up-regulated the immediate early gene IEX-1S
through an NF–B-dependent mechanism in breast cancer cells, CELL BIOLOGY and IEX-1S, when overexpressed in these cells, inhibited growth Abbreviations: MIS, Mullerian-inhibiting substance; rhMIS, human recombinant MIS; (4), indicating a negative growth regulatory role for this newly TGF-, transforming growth factor ; DHT, 5␣-dihydrotestosterone; EMSA, electrophoretic mobility-shift assay. identified NF–B-inducible gene. MIS type II receptor expression in the Mullerian duct is *D.L.S. and Y.H. contributed equally to this work. temporally and spatially regulated in the male embryo (11). †To whom reprint requests should be addressed at: Department of Pediatric Surgery, WRN 1024, Massachusetts General Hospital, Boston, MA 02114. E-mail: maheswaran@ Receptor expression is initially highest at the cranial end and helix.mgh.harvard.edu. gradually shifts to the caudal region, a pattern recapitulated by The publication costs of this article were defrayed in part by page charge payment. This the apoptotic index along the duct. The most distal ends of the article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Mullerian duct that express the MIS type II receptor persist in §1734 solely to indicate this fact.
www.pnas.org͞cgi͞doi͞10.1073͞pnas.221599298 PNAS ͉ January 8, 2002 ͉ vol. 99 ͉ no. 1 ͉ 239–244 Downloaded by guest on September 24, 2021 Fig. 1. MIS type II receptor expression in the prostate. (A) Receptor expression in the developing Mullerian duct of 16-day whole male rat embryos was analyzed by in situ hybridization by using antisense (Left) and sense (Right) rat MIS type II receptor probes. Open arrows demonstrate receptor expression in the ducts distal to the testicular end, and the closed arrow demonstrates expression in the area where the ducts fuse to form the prostatic utricle. T indicates the testes. (B) MIS type II receptor mRNA expression in the mouse prostate gland. Total RNA (100 g) isolated from mouse prostate was analyzed by RNase protection assay, and 100 g of yeast tRNA was hybridized with the probe and incubated with or without RNase to test the activity of RNases and probe integrity, respectively. Five micrograms of total RNA from mouse testis was analyzed as a positive control. Positions of the full-length probe and protected MIS type II receptor fragment are indicated. (C) Expression of MIS type II receptor in the human prostate. cDNA generated from prostate tissue excised from two prostate cancer patients was analyzed by reverse transcription–PCR by using primers specific for exons 1 and 5. A DNA fragment of the expected size (582 bp) is shown. M, 100-bp marker. (D) MIS type II receptor protein expression in human prostate cancer cell lines. Total protein (100 g) from LNCaP, DU-145, and PC3 cells was analyzed by Western blot. A parallel blot was probed with the preimmune rabbit serum. The position of the MIS type II receptor is shown. (E) Expression of ALK2 and ALK 6 type I receptors in human prostate cancer cells. cDNA generated from LNCaP and PC3 cells were analyzed by reverse transcription–PCR. Closed arrows indicate DNA fragments of the expected sizes (283 bp for ALK2 and 202 bp for ALK6). M, 100-bp marker.
PCR Analyses. ALK2 and ALK6 were amplified by PCR from the hospital. All experiments were performed by using ket- cDNAs derived from LNCaP and PC3 cells by using the follow- amine͞xylazine (100͞10 mg͞kg) for anesthesia. Each animal ing primers: ALK2, sense 5Ј-AAACCAGCCATTGCCCATCG- was injected i.p. with 100 g of rhMIS or PBS. Blood was drawn 3Ј; antisense 5Ј-TACCATTGCTCACCATCCGC-3Ј. The DNA from the animals at the time of tissue harvest to determine the fragment amplified by these primers spans amino acids 327–422 circulating level of rhMIS by using an MIS-ELISA. of the ALK2 protein. ALK6 was amplified by using the following primers: sense 5Ј-ATGCTTTTGCGAAGTGCAGG-3Ј; anti- Human Studies. Discarded human prostate tissue was obtained sense 5Ј-TGACCACAGGCAACCCAGAG-3Ј. The DNA frag- from the Massachusetts General Hospital tissue bank by using ment amplified by these primers spans the first 67 amino acids protocols approved by the Institutional Review Board-Human at the N terminus of the ALK6 protein. Research Committee of the hospital.
Antibodies and Western Blot Analyses. The rabbit MIS type II Results receptor antibody is described in ref. 4. Western analysis was MIS Type II Receptor Expression in the Prostate. As development performed as described (4). progresses, MIS type II receptor expression is more intense and persistent in the Mullerian duct mesenchyme most distal to the RNase Protection Assay. RNase protection assays and the ribo- testicular end and in the fused caudal ends that form the probes used to detect MIS type II receptor and IEX-1͞gly96 prostatic utricle (Fig. 1A). Thus, we tested the hypothesis that expression are described in ref. 5. adult prostate might express the MIS type II receptor and may be responsive to the MIS ligand. Animal Studies. To study the effects of rhMIS on the prostate MIS type II receptor mRNA expression in mouse prostate was gland, adult male C3H mice (8 weeks old; average weight 25 g) detected by RNase protection assay by using an antisense probe were obtained from the Edwin L. Steele Laboratory, Massachu- that contained exon 11 and the 3Ј untranslated region of the setts General Hospital, Boston, MA. All animals were cared for, mouse MIS type II receptor. Detection of a band of the expected and experiments were performed in this facility under Assess- size in the prostate, which comigrated with that from the testis, ment and Accreditation of Laboratory Animal Care-approved indicated that MIS type II receptor mRNA was expressed guidelines by using protocols approved by the Institutional in normal prostate but at a level lower than that in the testis Review Board-Institutional Animal Care and Use Committee of (Fig. 1B).
240 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.221599298 Segev et al. Downloaded by guest on September 24, 2021 Fig. 2. MIS induces NF-B DNA binding and IEX-1S expression in prostate cancer cells. Androgen-dependent LNCaP cells (A) and androgen-independent DU145 cells (B) were treated with 35 nM MIS, and 3 g of nuclear proteins were analyzed by EMSA by using a 32P-labeled NF-B oligonucleotide probe. Oligonucleotide competition was done with 50-fold excess of cold NF-B oligo by using the sample treated with MIS for 1 h. (A and B, Right) Antibody supershift experiments were done with samples treated with MIS for 1 h. The position of the NF-B DNA protein complex (closed arrow) and antibody supershifted complexes (open arrows) are indicated. * represents the most rapidly migrating complex that is blocked with excess unlabeled oligonucleotide but remains unchanged with MIS treatment. (C) MIS induces the expression of IEX-1. LNCaP (Left) and DU145 (Right) cells were treated with 35 nM MIS, and 10 g of total RNA was analyzed by Northern blot. Hybridization to 18S rRNA is shown to control for loading. (D Left) Total RNA (40 g) isolated from untreated and MIS (2 h)-treated LNCaP cells was analyzed by RNase protection assay. Positions of the probe and the protected fragments resulting from exons 1 and 2 of IEX-1S mRNA are indicated. (Right) Schematic representation of the human IEX-1L antisense riboprobe used for RNase protection assay. (E) MIS induced IEX-1 requires degradation of IB␣.(Upper) Vector or IB␣DN-transfected LNCaP cells were treated with 35 nM rhMIS for 1 h, and NF-B binding activity was analyzed by EMSA. (Lower) OCT-1 DNA binding was analyzed to ensure that equal amount of protein was analyzed and to demonstrate the specificity of NF-B induction by MIS. (F Upper) Vector or IB␣DN-transfected LNCaP cells were treated with MIS for 0 and 3 h; 10 g of total cellular RNA was analyzed for induction of IEX-1. (Lower) Hybridization to glyceraldehyde-3-phosphate dehydrogenase is shown as control for loading.
MIS type II receptor was also detected in human prostate MIS Activates NF-B Signaling in Prostate Cancer Cells. As with breast tissue obtained from patients with prostate carcinoma by reverse cancer cells (4), MIS induced NF-B DNA binding in prostate transcription–PCR (Fig. 1C). The DNA sequence of the frag- cancer cells. Supershift experiments demonstrated that the ment was identical to that of exons 1–5 of the human MIS type complex consisted of p50 and p65 subunits (Fig. 2A). Up- II receptor (data not shown). regulation of NF-B DNA binding activity composed of p50, p65 Immunoblot analysis of total cellular protein isolated from subunits after treatment with MIS was also observed in andro- LNCaP, DU-145, and PC-3 cells by using protein A-purified gen-independent DU145 cells (Fig. 2B). Similar results were rabbit MIS type II receptor antibody (4, 14, 15) demonstrated the obtained with PC3 cells (data not shown). presence of the 63-kDa endogenous MIS type II receptor We had previously shown that activation of NF-B in breast CELL BIOLOGY protein. Antibody specificity was shown with rabbit preimmune cancer cells after MIS treatment led to the induction of IEX-1 (4, serum (Fig. 1D). 5), an NF-B-inducible immediate early gene. PRG1 and gly96 To determine whether ALK2 and ALK6 type I receptors represent the rat and mouse homologues, respectively, of IEX-1 (6–8) are potentially involved in MIS signaling in the prostate, (17). Consistent with the up-regulation of NF-B DNA binding expression of ALK2 and ALK6 in prostate cancer cells was activity, MIS increased the expression of IEX-1 mRNA in verified by reverse transcription–PCR. Both LNCaP and PC3 LNCaP and DU145 cells (Fig. 2C). cells expressed ALK2 and ALK6 transcripts (Fig. 1E). The The IEX-1 gene encodes two proteins, IEX-1S and IEX-1L, presence of ALK2 in LNCaP cells was previously demonstrated which arise from two splice variants. RNase protection assay by PCR amplification and Western blot analysis (16). using an antisense riboprobe specific for human IEX-1L
Segev et al. PNAS ͉ January 8, 2002 ͉ vol. 99 ͉ no. 1 ͉ 241 Downloaded by guest on September 24, 2021 Fig. 3. Androgen activates NF-B DNA binding but does not induce IEX-1 mRNA. (A) LNCaP cells grown in androgen-deprived medium for 5 days were treated with 10 nM DHT; 3 g of nuclear proteins was analyzed by EMSA by using an NF-B oligonucleotide probe. Closed arrows indicate the position of the NF-B͞DNA protein complexes. Antibody supershift experiments were done by addition of anti-p65 and anti-p50 antibodies to the binding reaction. (B) LNCaP cells were grown in androgen-depleted medium for 2 days and treated with 10 nM DHT, 10 nM DHT, and 35 nM MIS, or 35 nM MIS alone. IEX-1 expression was analyzed by Northern blot. Hybridization to 18S rRNA is shown to control for loading.
demonstrated two protected fragments corresponding to exon 1 There was also a minor, faster migrating DNA͞protein complex, (211 nt) and exon 2 (261 nt), suggesting that IEX-1S was which consisted of p50 homodimers. the predominant transcript expressed after treatment with MIS Although both MIS and DHT induced NF-B DNA binding (Fig. 2D). activity in LNCaP cells, DHT did not influence IEX-1 mRNA To investigate whether induction of IEX-1S mRNA required expression; MIS induced IEX-1 expression in LNCaP cells, degradation of IB␣, we generated LNCaP cell clones that were whereas DHT did not (Fig. 3B). However, DHT slightly dimin- stably transfected with dominant negative IB␣ (IB␣DN). ished MIS-mediated induction of IEX-1 (Fig. 3B). These results Because the phosphorylation sites of wild-type IB␣, serines 32 indicate that MIS-induced activation of the NF- B pathway in and 36, are converted to alanines in the dominant negative prostate cancer cells does not require androgen signaling and protein, it cannot be phosphorylated or targeted for degradation that the type of stimulus seems to be a critical factor in determining the pattern of gene expression elicited after acti- (18). Thus, it functions as a superrepressor of NF-B activation. vation of NF-B. As reported previously (4), NF-B activation after exposure to MIS was abrogated in IB␣DN-expressing cells compared with MIS Induces IEX-1S mRNA in Prostate Glands of Mice in Vivo. We next those transfected with vector (Fig. 2E Upper). IEX-1S mRNA tested whether exposure to MIS can result in IEX-1 induction in was induced by MIS in LNCaP cells transfected with vector but ͞ ␣ vivo. MIS induced gly96 IEX-1 expression in the prostate glands not in cells expressing I B DN, suggesting that an increase in of mice (n ϭ 3) compared with PBS-injected controls (n ϭ 3, Fig. IEX-1S by MIS required activation of NF- B DNA binding 4A Left). PhosphorImaging of band intensities and normaliza- activity (Fig. 2F). tion to 18S rRNA demonstrated Ϸ3-fold increase in IEX-1 expression after6hofexposure to MIS (Fig. 4A Right). The Androgen Induces NF-B DNA Binding but Not IEX-1 mRNA. We next levels of circulating rhMIS in the injected animals were esti- determined whether androgen regulates NF-B signaling and mated to be 2–4 g͞ml by MIS-ELISA (20). Consistent with IEX-1 expression in prostate cancer cells. Consistent with a results in human prostate cancer cells, RNase protection assay report by Ripple et al. (19), DHT induced an NF-B complex demonstrated that MIS predominantly induced the gly96S͞ containing p50͞p65 heterodimers in LNCaP cells (Fig. 3A). IEX-1S transcript in the prostate gland in vivo (Fig. 4B).
Fig. 4. MIS induces IEX-1 expression in the prostate gland of mice in vivo.(A Left) Prostate glands of adult male mice were harvested 6 h after injecting 100 gofMIS͞animal, and total RNA was analyzed for gly96͞IEX-1 expression. RNA isolated from the prostate of mice 6 h after injecting PBS was used as control. Hybridization to mouse 18S rRNA is shown to control for loading. (Right) To quantify the changes in gly96͞IEX-1 expression in the prostate, the bands were quantified by using a PhosphorImager and IQMAC data analysis software. The differences in gly96͞IEX-1 mRNA intensities between PBS and MIS-treated samples were statistically significant (P ϭ 0.0005) as defined by unpaired Student’s t test. (B) Total RNA (10 g) from the prostate glands of mice injected with MIS was analyzed by RNase protection assay by using a mouse gly96͞IEX-1S antisense riboprobe. Equal amount of yeast tRNA was hybridized with the probe and incubated with or without RNase to test the activity of RNases and probe integrity, respectively. Positions of the probe and the protected fragment that results from the mouse gly96͞IEX-1S transcript are indicated.
242 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.221599298 Segev et al. Downloaded by guest on September 24, 2021 receptor null mice (23, 24) and MIS overproducing transgenic mice (25). The MIS type II receptor null male mice have persistent Mullerian ducts, a phenotype resembling that of MIS ligand-deficient mice (23, 24). Male mice overproducing MIS have undescended testes that become depleted of germ cells, lack seminal vesicles, and have underdeveloped epididymides and low levels of serum testosterone (25), suggesting a pivotal role for MIS in androgen biosynthesis. Injecting MIS into luteinizing hormone-stimulated adult male rats and mice de- creases the testosterone in serum and testicular extracts (26). MIS suppresses androgen synthesis by inhibiting the mRNA that encodes P450c17 hydroxylase͞lyase, the enzyme responsible for the conversion of progesterone to androstenedione (27). Thus, MIS may play a key role in regulating the growth and differen- tiation of tissues, such as the prostate gland, that depend on androgen for these processes. Our results demonstrate that MIS may also regulate intracellular growth-regulatory pathways in the prostate (Fig. 5B). Signal transduction by the TGF- superfamily is mediated through receptor-activated Smad proteins as well as other dis- tinct pathways such as induction of the mitogen-activated protein kinase cascade initiated by TAK1 (TGF--activated kinase-1) (28). Although heterodimerization of either ALK2 or ALK6 to the MIS type II receptor induces phosphorylation of the Smad1 protein, induction of NF-B and IEX-1S by MIS is likely to be Fig. 5. Stable expression of IB␣DN in LNCaP cells abrogates MIS-mediated independent of MIS-mediated Smad1 activation, as expression inhibition of growth. (A) Vector or IB␣DN-transfected LNCaP cells were of IB␣DN in LNCaP cells completely abolishes these events. treated with 35 nM rhMIS for 4 days, and cell numbers were calculated by Activation of NF-B by external stimuli in most cell systems using a Coulter counter (n ϭ 3). The mean number of cells in the untreated plates was set at 100%. (B) Model for MIS-mediated regulation of prostate involves a classic mechanism that requires degradation of I B (9, 10). However, induction of the HIV 1 enhancer by TGF-- cancer cell growth. MIS, synthesized by Sertoli cells of the testis, in addition to inhibiting the growth of Leydig cells also blocks the production of testoster- stimulated NF- B occurs through a mechanism that does not one, a key regulator of prostate growth. Furthermore, MIS also initiates an entail degradation of IB (29). The ablation of MIS-mediated androgen-independent intracellular signaling cascade (e.g., induction of NF- activation of NF-B signaling by IB␣DN suggests that degra- B) and antagonizes androgen-induced growth-regulatory pathways such as dation of IB is required for this process in prostate cancer cells. a decrease in p16 expression and an increase in hyperphosphorylated retino- Although TAK1 has been implicated in the activation of NF-B blastoma protein (pRB). DNA binding activity induced by TGF- (30), the kinase(s) responsible for MIS-induced phosphorylation of IB remain(s) to be identified. MIS Inhibits LNCaP Cell Growth Through an NF-B-Dependent Path- NF-B can exert either a proapoptotic or antiapoptotic signal way. To study the effect of MIS on prostate cancer cell growth, ␣ depending on the cell type and stimulus (9, 10). Interestingly, vector and I B DN-transfected LNCaP cells were treated with induction of NF-B can also induce or block apoptosis within the MIS. MIS inhibited the growth of LNCaP cells by 50%, and same cell in many cell types (31, 32). For example, expression of ablation of NF-B activation impeded its growth-inhibitory ␣ the more stable mutant form of I B in the prostate carcinoma effects (Fig. 5A), suggesting that NF- B activation was required cell line AT3 protects cells from hydrogen peroxide and Sindbis for MIS-mediated growth inhibition. virus-induced apoptosis, but it promotes tumor necrosis factor ␣ Discussion and staurosporine-induced apoptosis (31). Although the precise mechanism for this dual role of NF-B is not yet clear, differ- Although high levels of MIS type II receptor mRNA have been ential regulation of genes that control cell cycle progression, previously reported in the testis and ovary (2), analyses of other apoptosis, and cell survival, and interaction with other effectors tissues reveal lower levels of receptor in the mammary gland and of cell growth͞death may by involved in directing the cells to fetal rat lungs (2–5). We now demonstrate that both the prostate either survive or die. Indeed, both DHT and MIS induced NF-B gland and prostate cancer cells express the MIS type II receptor complexes in LNCaP cells, but androgen, unlike MIS, did not as well as putative MIS type I receptors. Induction of NF- B increase IEX-1 expression, demonstrating the strict dependence signaling in human prostate cancer cells, and inhibition of of gene expression on the stimulating agent. prostate cancer cell growth by MIS through an NF- B- The absence of IEX-1 induction in LNCaP cells by androgen dependent manner suggest that MIS may have a physiological suggests that up-regulation of IEX-1S mRNA by MIS occurs role in regulating the growth of the prostate gland. The presence through a mechanism that does not involve androgen-induced of MIS in the human seminal vesicle fluid at significantly higher signaling. However, preliminary results suggest that MIS can
concentrations (150 pM) than that observed in adult serum (11 overcome androgen-mediated repression of the cyclin- CELL BIOLOGY pM) (21) also makes a physiological role for MIS in prostate dependent kinase inhibitor p16 and androgen-induced hyper- growth very likely. Although the concentration of MIS (35 nM) phosphorylation of the retinoblastoma tumor suppressor gene required for in vitro experiments is about 200-fold higher than (pRb) in LNCaP cells (T.T.T. and S.M., unpublished observa- the circulating levels of MIS in the serum or that present in the tion). Thus, in addition to the effect of MIS on NF-Binthe seminal fluid, it is consistent with the concentration of MIS prostate, MIS may perturb other pathways affected by androgen required to cause regression of the Mullerian duct in organ that regulate prostate growth (Fig. 5B). Similar results have been culture assays (7, 11, 22). shown for activins and TGF-, both of which can inhibit the The importance of MIS-mediated signaling in male sexual growth of LNCaP cells in the absence or presence of stimulation development was demonstrated in MIS ligand and type II with androgen (33–39).
Segev et al. PNAS ͉ January 8, 2002 ͉ vol. 99 ͉ no. 1 ͉ 243 Downloaded by guest on September 24, 2021 Human prostate tumors and cancer cells express ligands and MIS would be of potential therapeutic benefit in treatment of receptors belonging to the TGF- superfamily. TGF- recep- prostate cancer. tors, however, are frequently lost in prostate cancer cells, which consequently acquire resistance to the apoptotic effect of TGF- We thank Drs. James Lorenzen, Jose Teixeira, and Trent Clarke for (40). BMP-2 receptors and activin and inhibin are also present critically reading this manuscript. We also thank Drs. Wylie Vale and in prostate tissue (41, 42). Prostate tumors express significantly Alan Schneyer for comprehensive external review of this manuscript. lower levels of activin receptor 1B mRNA when compared with This work was supported by the National Institutes of Health, National nonmalignant tissue (43). Both activin and TGF- inhibit pro- Cancer Institute Training Grant in Cancer Biology F32 CA77945-01A1, liferation and induce apoptosis of human prostatic cancer cell and a Resident Research Award from the American College of Surgeons lines (33–39). Despite the fact that MIS belongs to this group of (to D.L.S.), a fellowship fund from the Department of Surgery, Massa- polypeptides and plays an important role in androgen biosyn- chusetts General Hospital (to Y.H.), National Institutes of Health thesis, its role in development and differentiation of the prostate Training Grant T32 CA-71345-04 and Marshall K. Bartlett Fellowship gland has not been investigated, although it is known that growth from the Massachusetts General Hospital Department of Surgery (to A.E.S.), and Grants HD32112 and CA17393 from the National Institutes of the prostate developmentally occurs at puberty when MIS of Health͞National Institute of Child Health and Human Development levels decline. The presence of MIS type II receptor in the and National Institutes of Health͞National Cancer Institute, respec- prostate and initiation of growth-regulatory pathways by MIS in tively (to P.K.D.), and by the Breast Cancer Research Grant from the prostate cancer cells and the prostate in vivo suggest that the Massachusetts Department of Public Health, the Harvard Medical prostate may be a target tissue for MIS action. Further charac- School 50th Anniversary Scholars in Medicine Award, the Avon Breast terization of the functional significance of the presence of the Cancer Pilot Project Grant, the Claflin Distinguished Scholar Award, MIS receptor in the prostate depends on establishing animal partial support from the Dana–Farber Harvard Breast Cancer Special- models to study the effect of MIS on prostate carcinoma cell ized Program of Research Excellence, and from National Institutes of growth in vivo; such studies will also help to determine whether Health͞National Cancer Institute Grant CA89138-01A1 (to S.M.).
1. Teixeira, J. & Donahoe, P. K. (1996) J. Androl. 17, 336–341. 22. Roberts, L. M., Hirokawa, Y., Nachtigal, M. W. & Ingraham, H. A. (1999) Dev. 2. Teixeira, J., He, W. W., Shah, P. C., Morikawa, N., Lee, M. M., Catlin, E. A., Biol. 208, 110–122. Hudson, P. L., Wing, J., Maclaughlin, D. T. & Donahoe, P. K. (1996) 23. Behringer, R. R., Finegold, M. J. & Cate, R. L. (1994) Cell 79, 415–425. Endocrinology 137, 160–165. 24. Mishina, Y., Rey, R., Finegold, M. J., Matzuk, M. M., Josso, N., Cate, R. L. & 3. Catlin, E. A., Tonnu, V. C., Ebb, R. G., Pacheco, B. A., Manganaro, T. F., Behringer, R. R. (1996) Genes Dev. 10, 2577–2587. Ezzell, R. M., Donahoe, P. K. & Teixeira, J. (1997) Endocrinology 138, 790–796. 25. Behringer, R. R., Cate, R. L., Froelick, G. J., Palmiter, R. D. & Brinster, R. L. 4. Segev, D. L., Ha, T. U., Tran, T. T., Kenneally, M., Harkin, P., Jung, M., (1990) Nature (London) 345, 167–170. MacLaughlin, D. T., Donahoe, P. K. & Maheswaran, S. (2000) J. Biol. Chem. 26. Trbovich, A. M., Sluss, P. M., Laurich, V. M., O’Neill, F. H., MacLaughlin, 275, 28371–28379. D. T., Donahoe, P. K. & Teixeira, J. (2001) Proc. Natl. Acad. Sci. USA 98, 5. Segev, D. L., Hoshiya, Y., Stephen, A. E., Hoshiya, M., Tran, T. T., MacLaugh- 3393–3397. (First Published March 6, 2001; 10.1073͞pnas.051632298) lin, D. T., Donahoe, P. K. & Maheswaran, S. (2001) J. Biol. Chem. 276, 27. Teixeira, J., Fynn-Thompson, E., Payne, A. H. & Donahoe, P. K. (1999) 26799–26806. Endocrinology 140, 4732–4738. 6. Clarke, T. R., Hoshiya, Y., Yi, S. E., Liu, X., Lyons, K. M. & Donahoe, P. K. 28. Yamaguchi, K., Shirakabe, K., Shibuya, H., Irie, K., Oishi, I., Ueno, N., (2001) Mol. Endocrinol. 15, 946–959. Taniguchi, T., Nishida, E. & Matsumoto, K. (1995) Science 270, 2008–2011. 7. Gouedard, L., Chen, Y. G., Thevenet, L., Racine, C., Borie, S., Lamarre, I., 29. Li, J. M., Shen, X., Hu, P. P. & Wang, X. F. (1998) Mol. Cell. Biol. 18, 110–121. Josso, N., Massague, J. & di Clemente, N. (2000) J. Biol. Chem. 275, 30. Sakurai, H., Shigemori, N., Hasegawa, K. & Sugita, T. (1998) Biochem. Biophys. 27973–27978. Res. Commun. 243, 545–549. 8. Visser, J. A., Olaso, R., Verhoef-Post, M., Kramer, P., Themmen, A. P. & 31. Lin, K. I., DiDonato, J. A., Hoffmann, A., Hardwick, J. M. & Ratan, R. R. Ingraham, H. A. (2001) Mol. Endocrinol. 15, 936–945. (1998) J. Cell Biol. 141, 1479–1487. 9. Baichwal, V. R. & Baeuerle, P. A. (1997) Curr. Biol. 7, R94–R96. 32. Lin, B., Williams-Skipp, C., Tao, Y., Schleicher, M. S., Cano, L. L., Duke, R. C. 10. Barkett, M. & Gilmore, T. D. (1999) Oncogene 18, 6910–6924. & Scheinman, R. I. (1999) Cell Death Differ. 6, 570–582. 11. Allard, S., Adin, P., Gouedard, L., di Clemente, N., Josso, N., Orgebin-Crist, 33. Bruckheimer, E. M. & Kyprianou, N. (2001) Endocrinology 142, 2419–2426. M. C., Picard, J. Y. & Xavier, F. (2000) Development (Cambridge, U.K.) 127, 34. Dalkin, A. C., Gilrain, J. T., Bradshaw, D. & Myers, C. E. (1996) Endocrinology 3349–3360. 137, 5230–5235. 12. Carlson, B. M. (1999) Human Embryology and Developmental Biology, 2nd Ed. 35. Hayes, S. A., Zarnegar, M., Sharma, M., Yang, F., Peehl, D. M., ten Dijke, P. 13. Ragin, R. C., Donahoe, P. K., Kenneally, M. K., Ahmad, M. F. & MacLaughlin, & Sun, Z. (2001) Cancer Res. 61, 2112–2118. D. T. (1992) Protein Expression Purif. 3, 236–245. 36. Kang, H. Y., Lin, H. K., Hu, Y. C., Yeh, S., Huang, K. E. & Chang, C. (2001) 14. Ha, T. U., Segev, D. L., Barbie, D., Masiakos, P. T., Tran, T. T., Dombkowski, Proc. Natl. Acad. Sci. USA 98, 3018–3023. (First Published March 6, 2001; D., Glander, M., Clarke, T. R., Lorenzo, H. K., Donahoe, P. K. & Maheswaran, 10.1073͞pnas.061305498) S. (2000) J. Biol. Chem. 275, 37101–37109. 37. Kim, I. Y., Zelner, D. J., Sensibar, J. A., Ahn, H. J., Park, L., Kim, J. H. & Lee, 15. Masiakos, P. T., MacLaughlin, D. T., Maheswaran, S., Teixeira, J., Fuller, A. F., C. (1996) Exp. Cell Res. 222, 103–110. Jr., Shah, P. C., Kehas, D. J., Kenneally, M. K., Dombkowski, D. M., Ha, T. U., 38. McPherson, S. J., Thomas, T. Z., Wang, H., Gurusinghe, C. J. & Risbridger, et al. (1999) Clin. Cancer Res. 5, 3488–3499. G. P. (1997) J. Endocrinol. 154, 535–545. 16. Kim, I. Y., Zelner, D. J. & Lee, C. (1998) Exp. Cell Res. 241, 151–160. 39. Wang, Q. F., Tilly, K. I., Tilly, J. L., Preffer, F., Schneyer, A. L., Crowley, W. F., 17. Schafer, H., Diebel, J., Arlt, A., Trauzold, A. & Schmidt, W. E. (1998) FEBS Jr. & Sluss, P. M. (1996) Endocrinology 137, 5476–5483. Lett. 436, 139–143. 40. Wikstrom, P., Bergh, A. & Damber, J. E. (2000) Scand. J. Urol. Nephrol. 34, 18. Brown, K., Gerstberger, S., Carlson, L., Franzoso, G. & Siebenlist, U. (1995) 85–94. Science 267, 1485–1488. 41. Iwasaki, S., Tsuruoka, N., Hattori, A., Sato, M., Tsujimoto, M. & Kohno, M. 19. Ripple, M. O., Henry, W. F., Schwarze, S. R., Wilding, G. & Weindruch, R. (1995) J. Biol. Chem. 270, 5476–5482. (1999) J. Natl. Cancer Inst. 91, 1227–1232. 42. Risbridger, G. P., Thomas, T., Gurusinghe, C. J. & McFarlane, J. R. (1996) J. 20. Hudson, P. L., Dougas, I., Donahoe, P. K., Cate, R. L., Epstein, J., Pepinsky, Endocrinol. 149, 93–99. R. B. & MacLaughlin, D. T. (1990) J. Clin. Endocrinol. Metab. 70, 16–22. 43. van Schaik, R. H., Wierikx, C. D., Timmerman, M. A., Oomen, M. H., van 21. Fenichel, P., Rey, R., Poggioli, S., Donzeau, M., Chevallier, D. & Pointis, G. Weerden, W. M., van der Kwast, T. H., van Steenbrugge, G. J. & de Jong, F. H. (1999) Hum. Reprod. 14, 2020–2024. (2000) Br. J. Cancer 82, 112–117.
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