Mullerian-inhibiting substance regulates NF-kappa B signaling in the prostate in vitro and in vivo

Abstract

Mullerian-inhibiting substance (MIS) is a member of the transforming growth factor beta 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-kappaB 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 IkappaBalpha expression ablated both MIS-induced increase of IEX-1S mRNA and inhibition of growth, indicating that activation of NF-kappaB signaling was required for these processes. Androgen also induced NF-kappaB 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-kappaB signaling and growth in the prostate gland.

Figure 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 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.

Figure 2

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.

Figure 3

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.

Figure 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 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.

Figure 5

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).