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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2002 Jan 2;99(1):239–244. doi: 10.1073/pnas.221599298

Mullerian-inhibiting substance regulates NF–κB signaling in the prostate in vitro and in vivo

Dorry L Segev 1,*, Yasunori Hoshiya 1,*, Makiko Hoshiya 1, Trinh T Tran 1, Jennifer L Carey 1, Antonia E Stephen 1, David T MacLaughlin 1, Patricia K Donahoe 1, Shyamala Maheswaran 1,†
PMCID: PMC117545  PMID: 11773638

Abstract

Mullerian-inhibiting substance (MIS) is a member of the transforming growth factor β superfamily, a class of molecules that regulates growth, differentiation, and apoptosis in many cells. MIS type II receptor in the Mullerian duct is temporally and spatially regulated during development and becomes restricted to the most caudal ends that fuse to form the prostatic utricle. In this article, we have demonstrated MIS type II receptor expression in the normal prostate, human prostate cancer cell lines, and tissue derived from patients with prostate adenocarcinomas. MIS induced NF–κB DNA binding activity and selectively up-regulated the immediate early gene IEX-1S in both androgen-dependent and independent human prostate cancer cells in vitro. Dominant negative IκBα expression ablated both MIS-induced increase of IEX-1S mRNA and inhibition of growth, indicating that activation of NF–κB signaling was required for these processes. Androgen also induced NF–κB DNA binding activity in prostate cancer cells but without induction of IEX-1S mRNA, suggesting that MIS-mediated increase in IEX-1S was independent of androgen-mediated signaling. Administration of MIS to male mice induced IEX-1S mRNA in the prostate in vivo, suggesting that MIS may function as an endogenous hormonal regulator of NF–κB signaling and growth in the prostate gland.

Keywords: IEX-1

Mullerian-inhibiting substance (MIS) belongs to the transforming growth factor β (TGF-β) superfamily, which also includes activins, inhibins, and the bone morphogenetic proteins. The Mullerian duct, the anlagen of the uterus, Fallopian tubes, and upper vagina in the female, regresses in the presence of MIS in male embryos (1). The MIS type II receptor, a transmembrane serine threonine kinase, is expressed at high levels in the Mullerian duct and in the embryonic and adult gonads (2). Lower levels of receptor expression were detected in the mammary gland and embryonic rat lungs (3–5), which show functional responses to MIS.

The binding of MIS to its receptor initiates a signaling cascade that depends on the recruitment of type I receptors ALK2 and ALK6 (6–8). We recently demonstrated that MIS inhibited the growth of breast cancer cells in vitro through induction of NF–κB (4), a family of transcription factors that includes p50, p65, p52, and c-rel. These transactivators form either homodimers or heterodimers that are retained in the cytoplasm by a class of inhibitor proteins, IκB-α, IκB-β, IκB-γ, and IκB-ɛ. Nuclear translocation of NF–κB after phosphorylation and degradation of IκB results in changes in gene expression (9, 10). MIS selectively up-regulated the immediate early gene IEX-1S through an NF–κB-dependent mechanism in breast cancer cells, and IEX-1S, when overexpressed in these cells, inhibited growth (4), indicating a negative growth regulatory role for this newly identified NF–κB-inducible gene.

MIS type II receptor expression in the Mullerian duct is temporally and spatially regulated in the male embryo (11). Receptor expression is initially highest at the cranial end and gradually shifts to the caudal region, a pattern recapitulated by the apoptotic index along the duct. The most distal ends of the Mullerian duct that express the MIS type II receptor persist in the male as the prostatic utricle, around which the prostate forms later in development (12). Based on these observations, we tested the hypothesis that MIS type II receptor may be expressed in the prostate gland and that prostate may be an additional target tissue for the action of MIS. In this article, we demonstrate MIS type II receptor expression in the prostate glands of adult mice and human prostate tissue and cancer cell lines. As with breast cancer cells, MIS induced NF-κB signaling and the immediate early gene IEX-1S in prostate cancer cells and murine prostate glands in vivo. Abrogation of NF-κB activity with dominant negative IκB-α ablated MIS-mediated molecular events and inhibition of prostate cancer cell growth, suggesting that the prostate is a likely target for the action of MIS.

Materials and Methods

Cell Lines, Reagents, and Growth Inhibition Assays.

LNCaP cells were grown in RPMI 1640 containing 10% FBS, glutamine, and penicillin/streptomycin. DU145 cells were grown in Eagle's minimum essential medium containing Earle's BSS/2 mM L-glutamine/1.0 mM sodium pyruvate/0.1 mM nonessential aminoacids/1.5 g/liter sodium bicarbonate/10% FBS and penicillin/streptomycin. PC3 cells were grown in Ham's F12K medium supplemented with 10% FBS/2 mM L-glutamine/1.5 g/liter sodium bicarbonate and penicillin/streptomycin. To measure inhibition of cell proliferation, cultures were grown in the presence or absence of 35 nM MIS for 4 days, and cell numbers were determined by using a Coulter counter. To determine the effects of androgen, cells were grown in medium containing 7% charcoal-stripped serum devoid of androgen (Gemini Biological Products, Calabasas, CA) for 2–5 days and treated with 10 nM 5α-dihydrotestosterone (DHT, Sigma).

Human recombinant MIS (rhMIS) was collected from growth media of Chinese hamster ovary cells transfected with the human MIS gene and purified as described (13).

In Situ Hybridization, Electrophoretic Mobility-Shift Assay (EMSA), and Northern Blot.

Timed pregnant rats were purchased from Harlan Breeders (Indianapolis), 16-day embryos were harvested, and in situ hybridization to detect MIS type II receptor was performed as described (6).

Nuclear proteins were harvested, and EMSA was performed as described (4). Total RNA isolated from cells was probed with radiolabeled IEX-1, glyceraldehyde-3-phosphate dehydrogenase, or 18S ribosomal RNA as described (4).

PCR Analyses.

ALK2 and ALK6 were amplified by PCR from cDNAs derived from LNCaP and PC3 cells by using the following primers: ALK2, sense 5′-AAACCAGCCATTGCCCATCG-3′; antisense 5′-TACCATTGCTCACCATCCGC-3′. The DNA fragment amplified by these primers spans amino acids 327–422 of the ALK2 protein. ALK6 was amplified by using the following primers: sense 5′-ATGCTTTTGCGAAGTGCAGG-3′; antisense 5′-TGACCACAGGCAACCCAGAG-3′. The DNA fragment amplified by these primers spans the first 67 amino acids at the N terminus of the ALK6 protein.

Antibodies and Western Blot Analyses.

The rabbit MIS type II receptor antibody is described in ref. 4. Western analysis was performed as described (4).

RNase Protection Assay.

RNase protection assays and the riboprobes used to detect MIS type II receptor and IEX-1/gly96 expression are described in ref. 5.

Animal Studies.

To study the effects of rhMIS on the prostate gland, adult male C3H mice (8 weeks old; average weight 25 g) were obtained from the Edwin L. Steele Laboratory, Massachusetts General Hospital, Boston, MA. All animals were cared for, and experiments were performed in this facility under Assessment and Accreditation of Laboratory Animal Care-approved guidelines by using protocols approved by the Institutional Review Board-Institutional Animal Care and Use Committee of the hospital. All experiments were performed by using ketamine/xylazine (100/10 mg/kg) for anesthesia. Each animal was injected i.p. with 100 μg of rhMIS or PBS. Blood was drawn from the animals at the time of tissue harvest to determine the circulating level of rhMIS by using an MIS-ELISA.

Human Studies.

Discarded human prostate tissue was obtained from the Massachusetts General Hospital tissue bank by using protocols approved by the Institutional Review Board-Human Research Committee of the hospital.

Results

MIS Type II Receptor Expression in the Prostate.

As development progresses, MIS type II receptor expression is more intense and persistent in the Mullerian duct mesenchyme most distal to the testicular end and in the fused caudal ends that form the prostatic utricle (Fig. 1A). Thus, we tested the hypothesis that adult prostate might express the MIS type II receptor and may be responsive to the MIS ligand.

Figure 1.

Figure 1

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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 μg) isolated from mouse prostate was analyzed by RNase protection assay, and 100 μg 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 μg) 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.

MIS type II receptor mRNA expression in mouse prostate was detected by RNase protection assay by using an antisense probe that contained exon 11 and the 3′ untranslated region of the mouse MIS type II receptor. Detection of a band of the expected size in the prostate, which comigrated with that from the testis, indicated that MIS type II receptor mRNA was expressed in normal prostate but at a level lower than that in the testis (Fig. 1B).

MIS type II receptor was also detected in human prostate tissue obtained from patients with prostate carcinoma by reverse transcription–PCR (Fig. 1C). The DNA sequence of the fragment was identical to that of exons 1–5 of the human MIS type II receptor (data not shown).

Immunoblot analysis of total cellular protein isolated from LNCaP, DU-145, and PC-3 cells by using protein A-purified rabbit MIS type II receptor antibody (4, 14, 15) demonstrated the presence of the 63-kDa endogenous MIS type II receptor protein. Antibody specificity was shown with rabbit preimmune serum (Fig. 1D).

To determine whether ALK2 and ALK6 type I receptors (6–8) are potentially involved in MIS signaling in the prostate, expression of ALK2 and ALK6 in prostate cancer cells was verified by reverse transcription–PCR. Both LNCaP and PC3 cells expressed ALK2 and ALK6 transcripts (Fig. 1E). The presence of ALK2 in LNCaP cells was previously demonstrated by PCR amplification and Western blot analysis (16).

MIS Activates NF-κB Signaling in Prostate Cancer Cells.

As with breast cancer cells (4), MIS induced NF-κB DNA binding in prostate cancer cells. Supershift experiments demonstrated that the complex consisted of p50 and p65 subunits (Fig. 2A). Up-regulation of NF-κB DNA binding activity composed of p50, p65 subunits after treatment with MIS was also observed in androgen-independent DU145 cells (Fig. 2B). Similar results were obtained with PC3 cells (data not shown).

Figure 2.

Figure 2

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MIS induces NF-κB 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 μg of nuclear proteins were analyzed by EMSA by using a 32P-labeled NF-κB oligonucleotide probe. Oligonucleotide competition was done with 50-fold excess of cold NF-κB 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-κB 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 μg of total RNA was analyzed by Northern blot. Hybridization to 18S rRNA is shown to control for loading. (D Left) Total RNA (40 μg) 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 IκBα. (Upper) Vector or IκBαDN-transfected LNCaP cells were treated with 35 nM rhMIS for 1 h, and NF-κB 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-κB induction by MIS. (F Upper) Vector or IκBαDN-transfected LNCaP cells were treated with MIS for 0 and 3 h; 10 μg of total cellular RNA was analyzed for induction of IEX-1. (Lower) Hybridization to glyceraldehyde-3-phosphate dehydrogenase is shown as control for loading.

We had previously shown that activation of NF-κB in breast cancer cells after MIS treatment led to the induction of IEX-1 (4, 5), an NF-κB-inducible immediate early gene. PRG1 and gly96 represent the rat and mouse homologues, respectively, of IEX-1 (17). Consistent with the up-regulation of NF-κB DNA binding activity, MIS increased the expression of IEX-1 mRNA in LNCaP and DU145 cells (Fig. 2C).

The IEX-1 gene encodes two proteins, IEX-1S and IEX-1L, which arise from two splice variants. RNase protection assay using an antisense riboprobe specific for human IEX-1L demonstrated two protected fragments corresponding to exon 1 (211 nt) and exon 2 (261 nt), suggesting that IEX-1S was the predominant transcript expressed after treatment with MIS (Fig. 2D).

To investigate whether induction of IEX-1S mRNA required degradation of IκBα, we generated LNCaP cell clones that were stably transfected with dominant negative IκBα (IκBαDN). Because the phosphorylation sites of wild-type IκBα, serines 32 and 36, are converted to alanines in the dominant negative protein, it cannot be phosphorylated or targeted for degradation (18). Thus, it functions as a superrepressor of NF-κB activation. As reported previously (4), NF-κB activation after exposure to MIS was abrogated in IκBαDN-expressing cells compared with those transfected with vector (Fig. 2E Upper). IEX-1S mRNA was induced by MIS in LNCaP cells transfected with vector but not in cells expressing IκBαDN, suggesting that an increase in IEX-1S by MIS required activation of NF-κB DNA binding activity (Fig. 2F).

Androgen Induces NF-κB DNA Binding but Not IEX-1 mRNA.

We next determined whether androgen regulates NF-κB signaling and IEX-1 expression in prostate cancer cells. Consistent with a report by Ripple et al. (19), DHT induced an NF-κB complex containing p50/p65 heterodimers in LNCaP cells (Fig. 3A). There was also a minor, faster migrating DNA/protein complex, which consisted of p50 homodimers.

Figure 3.

Figure 3

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Androgen activates NF-κB 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 μg of nuclear proteins was analyzed by EMSA by using an NF-κB oligonucleotide probe. Closed arrows indicate the position of the NF-κB/DNA 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.

Although both MIS and DHT induced NF-κB DNA binding activity in LNCaP cells, DHT did not influence IEX-1 mRNA expression; MIS induced IEX-1 expression in LNCaP cells, whereas DHT did not (Fig. 3B). However, DHT slightly diminished MIS-mediated induction of IEX-1 (Fig. 3B). These results indicate that MIS-induced activation of the NF-κB pathway in prostate cancer cells does not require androgen signaling and that the type of stimulus seems to be a critical factor in determining the pattern of gene expression elicited after activation of NF-κB.

MIS Induces IEX-1S mRNA in Prostate Glands of Mice in Vivo.

We next tested whether exposure to MIS can result in IEX-1 induction in vivo. MIS induced gly96/IEX-1 expression in the prostate glands of mice (n = 3) compared with PBS-injected controls (n = 3, Fig. 4A Left). PhosphorImaging of band intensities and normalization to 18S rRNA demonstrated ≈3-fold increase in IEX-1 expression after 6 h of exposure to MIS (Fig. 4A Right). The levels of circulating rhMIS in the injected animals were estimated to be 2–4 μg/ml by MIS-ELISA (20). Consistent with results in human prostate cancer cells, RNase protection assay demonstrated that MIS predominantly induced the gly96S/IEX-1S transcript in the prostate gland in vivo (Fig. 4B).

Figure 4.

Figure 4

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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 μg of MIS/animal, and total RNA was analyzed for gly96/IEX-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 gly96/IEX-1 expression in the prostate, the bands were quantified by using a PhosphorImager and iqmac data analysis software. The differences in gly96/IEX-1 mRNA intensities between PBS and MIS-treated samples were statistically significant (P = 0.0005) as defined by unpaired Student's t test. (B) Total RNA (10 μg) from the prostate glands of mice injected with MIS was analyzed by RNase protection assay by using a mouse gly96/IEX-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 gly96/IEX-1S transcript are indicated.

MIS Inhibits LNCaP Cell Growth Through an NF-κB-Dependent Pathway.

To study the effect of MIS on prostate cancer cell growth, vector and IκBαDN-transfected LNCaP cells were treated with MIS. MIS inhibited the growth of LNCaP cells by 50%, and ablation of NF-κB activation impeded its growth-inhibitory effects (Fig. 5A), suggesting that NF-κB activation was required for MIS-mediated growth inhibition.

Figure 5.

Figure 5

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Stable expression of IκBαDN in LNCaP cells abrogates MIS-mediated inhibition of growth. (A) Vector or IκBαDN-transfected LNCaP cells were treated with 35 nM rhMIS for 4 days, and cell numbers were calculated by using a Coulter counter (n = 3). The mean number of cells in the untreated plates was set at 100%. (B) Model for MIS-mediated regulation of prostate 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 testosterone, a key regulator of prostate growth. Furthermore, MIS also initiates an androgen-independent intracellular signaling cascade (e.g., induction of NF-κB) and antagonizes androgen-induced growth-regulatory pathways such as a decrease in p16 expression and an increase in hyperphosphorylated retinoblastoma protein (pRB).

Discussion

Although high levels of MIS type II receptor mRNA have been previously reported in the testis and ovary (2), analyses of other tissues reveal lower levels of receptor in the mammary gland and fetal rat lungs (2–5). We now demonstrate that both the prostate gland and prostate cancer cells express the MIS type II receptor as well as putative MIS type I receptors. Induction of NF-κB signaling in human prostate cancer cells, and inhibition of prostate cancer cell growth by MIS through an NF-κB-dependent manner suggest that MIS may have a physiological role in regulating the growth of the prostate gland. The presence of MIS in the human seminal vesicle fluid at significantly higher concentrations (150 pM) than that observed in adult serum (11 pM) (21) also makes a physiological role for MIS in prostate growth very likely. Although the concentration of MIS (35 nM) required for in vitro experiments is about 200-fold higher than the circulating levels of MIS in the serum or that present in the seminal fluid, it is consistent with the concentration of MIS required to cause regression of the Mullerian duct in organ culture assays (7, 11, 22).

The importance of MIS-mediated signaling in male sexual development was demonstrated in MIS ligand and type II 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 decreases the testosterone in serum and testicular extracts (26). MIS suppresses androgen synthesis by inhibiting the mRNA that encodes P450c17 hydroxylase/lyase, the enzyme responsible for the conversion of progesterone to androstenedione (27). Thus, MIS may play a key role in regulating the growth and differentiation 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-β superfamily is mediated through receptor-activated Smad proteins as well as other distinct pathways such as induction of the mitogen-activated protein kinase cascade initiated by TAK1 (TGF-β-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-κB and IEX-1S by MIS is likely to be independent of MIS-mediated Smad1 activation, as expression of IκBαDN in LNCaP cells completely abolishes these events. Activation of NF-κB by external stimuli in most cell systems involves a classic mechanism that requires degradation of IκB (9, 10). However, induction of the HIV 1 enhancer by TGF-β-stimulated NF-κB occurs through a mechanism that does not entail degradation of IκB (29). The ablation of MIS-mediated activation of NF-κB signaling by IκBαDN suggests that degradation of IκB is required for this process in prostate cancer cells. Although TAK1 has been implicated in the activation of NF-κB DNA binding activity induced by TGF-β (30), the kinase(s) responsible for MIS-induced phosphorylation of IκB remain(s) to be identified.

NF-κB can exert either a proapoptotic or antiapoptotic signal depending on the cell type and stimulus (9, 10). Interestingly, induction of NF-κB can also induce or block apoptosis within the same cell in many cell types (31, 32). For example, expression of the more stable mutant form of IκBα in the prostate carcinoma cell line AT3 protects cells from hydrogen peroxide and Sindbis virus-induced apoptosis, but it promotes tumor necrosis factor α and staurosporine-induced apoptosis (31). Although the precise mechanism for this dual role of NF-κB is not yet clear, differential regulation of genes that control cell cycle progression, apoptosis, and cell survival, and interaction with other effectors of cell growth/death may by involved in directing the cells to either survive or die. Indeed, both DHT and MIS induced NF-κB complexes in LNCaP cells, but androgen, unlike MIS, did not increase IEX-1 expression, demonstrating the strict dependence of gene expression on the stimulating agent.

The absence of IEX-1 induction in LNCaP cells by androgen suggests that up-regulation of IEX-1S mRNA by MIS occurs through a mechanism that does not involve androgen-induced signaling. However, preliminary results suggest that MIS can overcome androgen-mediated repression of the cyclin-dependent kinase inhibitor p16 and androgen-induced hyperphosphorylation of the retinoblastoma tumor suppressor gene (pRb) in LNCaP cells (T.T.T. and S.M., unpublished observation). Thus, in addition to the effect of MIS on NF-κB in the prostate, MIS may perturb other pathways affected by androgen that regulate prostate growth (Fig. 5B). Similar results have been shown for activins and TGF-β, both of which can inhibit the growth of LNCaP cells in the absence or presence of stimulation with androgen (33–39).

Human prostate tumors and cancer cells express ligands and receptors belonging to the TGF-β superfamily. TGF-β receptors, however, are frequently lost in prostate cancer cells, which consequently acquire resistance to the apoptotic effect of TGF-β (40). BMP-2 receptors and activin and inhibin are also present in prostate tissue (41, 42). Prostate tumors express significantly lower levels of activin receptor 1B mRNA when compared with nonmalignant tissue (43). Both activin and TGF-β inhibit proliferation and induce apoptosis of human prostatic cancer cell lines (33–39). Despite the fact that MIS belongs to this group of polypeptides and plays an important role in androgen biosynthesis, its role in development and differentiation of the prostate gland has not been investigated, although it is known that growth of the prostate developmentally occurs at puberty when MIS levels decline. The presence of MIS type II receptor in the prostate and initiation of growth-regulatory pathways by MIS in prostate cancer cells and the prostate in vivo suggest that the prostate may be a target tissue for MIS action. Further characterization of the functional significance of the presence of the MIS receptor in the prostate depends on establishing animal models to study the effect of MIS on prostate carcinoma cell growth in vivo; such studies will also help to determine whether MIS would be of potential therapeutic benefit in treatment of prostate cancer.

Acknowledgments

We thank Drs. James Lorenzen, Jose Teixeira, and Trent Clarke for critically reading this manuscript. We also thank Drs. Wylie Vale and Alan Schneyer for comprehensive external review of this manuscript. This work was supported by the National Institutes of Health, National Cancer Institute Training Grant in Cancer Biology F32 CA77945-01A1, and a Resident Research Award from the American College of Surgeons (to D.L.S.), a fellowship fund from the Department of Surgery, Massachusetts General Hospital (to Y.H.), National Institutes of Health Training Grant T32 CA-71345-04 and Marshall K. Bartlett Fellowship from the Massachusetts General Hospital Department of Surgery (to A.E.S.), and Grants HD32112 and CA17393 from the National Institutes of Health/National Institute of Child Health and Human Development and National Institutes of Health/National Cancer Institute, respectively (to P.K.D.), and by the Breast Cancer Research Grant from the Massachusetts Department of Public Health, the Harvard Medical School 50th Anniversary Scholars in Medicine Award, the Avon Breast Cancer Pilot Project Grant, the Claflin Distinguished Scholar Award, partial support from the Dana–Farber Harvard Breast Cancer Specialized Program of Research Excellence, and from National Institutes of Health/National Cancer Institute Grant CA89138-01A1 (to S.M.).

Abbreviations

MIS

Mullerian-inhibiting substance

rhMIS

human recombinant MIS

TGF-β

transforming growth factor β

DHT

5α-dihydrotestosterone

EMSA

electrophoretic mobility-shift assay

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