A role of inhibin as a tumor suppressor in Sertoli cells: down-regulation upon aging and repression by a viral oncogene

Abstract

Inhibin, a member of the TGF-b superfamily, is synthesized in the testis by Sertoli cells and exerts an endocrine regulatory function on pituitary hormone synthesis. A distinct local function has been proposed, negatively controlling cellular growth in the testis (tumor suppressor activity). A critical test for the identi®cation of a tumor suppressor is the reversal of transformed growth properties upon re-expression of the gene in tumor-derived cell lines. Sertoli cell-derived tumoral lines were previously established from tumors that develop in elderly transgenic males which express in the testis the large T antigen of polyoma virus. Both the tumors and the cells in culture exhibited reduced levels of the inhibin a subunit mRNA. Stable transfectants were generated, in which this subunit was expressed from a heterologous promoter. All of them exhibited a strict inhibition of growth at con¯uency. On the other hand, in addition to an aging-related decrease in inhibin synthesis, the a subunit gene was down regulated in vivo in cells expressing the viral protein. The conjunction of these two factors accounts for the age-related occurrence of testicular cancers in the transgenic model, again pointing to inhibin as a potent cell growth regulator in the seminiferous epithelium.

Gene Control in Germinal Differentiation: Rnf6, a Transcription Regulatory Protein in the Mouse Sertoli Cell

“…Expression of the gene under control of the wild-type promoter was twofold greater than the activity observed with the mutated promoter. Interestingly, coexpression of PyLT repressed the wild-type promoter to the same extent, comparable to the decrease in Inha expression in the PyLT transgenic mouse testis in vivo (11), and it had no effect on the residual expression of the mutant (Fig. 1).…”

“…The PyLT protein of polyomavirus is a transcriptional regulator with a binding site in the viral promoter consisting of a repeat of the G(A/G)GGC nucleotide sequence (GGC box). Repeated GGC boxes are also present in the promoter of the ␣ subunit of inhibin (Inha), and we formulated a hypothesis of competitive inhibition by PyLT of the binding of a positive regulatory protein in the same region of the promoter (11). In order to evaluate the role of the GGC boxes in the regulation of Inha expression, we generated a mutated promoter by replacement of the two GGGGC motifs with GGTGC sequences.…”

“…Total RNA (1 g) prepared with a Total RNA Isolation kit (Boehringer Mannheim) was reverse transcribed with murine leukemia virus reverse transcriptase in accordance with the supplier's instructions. PCR amplification was performed with Taq DNA polymerase (Boehringer Mannheim) with oligonucleotide primers for Inha and Hprt as previously described (11). For the Hprt internal controls, a sample was taken after only 5 min of reverse transcription.…”

“…Plasmid pPyLT1 codes for PyLT (29). Construction of Inha reporter plasmid pInh-lacZ was done as described previously (11). The full-length cDNA encoding the Rnf6 protein was amplified by reverse transcription-PCR performed on testis mRNA with primers RNF6am (5ЈCAGGATGGATCTGTCT AGATC3Ј) and RNF6av (5ЈAGCAGACCCCACCTCTCAC3Ј) and cloned into PCR cloning vector pGEM-T easy (Promega).…”

“…Twenty micrograms of total RNA from testes or from cells in culture was prepared and hybridized as previously described (11).…”