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The present study examines novel mechanisms that regulate levels of the RI alpha subunit of cAMP-dependent protein kinase. We found that RI alpha protein is induced threefold by 8-(4-chlorophenyl)thio-cAMP in hormone responsive rat Sertoli cells, while total RI alpha mRNA is not correspondingly induced. Two RI alpha mRNA isoforms with different 5' untranslated sequences (RI alpha 1a and RI alpha 1b) are produced from the RI alpha gene in Sertoli cells. Deletion/mutation analysis of the cAMP-response-element-containing promoter upstream of the RI alpha exon 1b revealed that while mutation of the cAMP response element had no effects on cAMP-mediated induction, a 73-bp region of the RI alpha exon 1b itself conferred a fivefold to eightfold induction of reporter activity to homologous and heterologous promoters. The responsiveness of this region was dependent on a sense orientation downstream of the promoter start sites and had no effect on reporter mRNA, indicating that the cAMP-mediated induction occurs at the post-transcriptional level. Modeling of the RI alpha 1b 5' UTR secondary structure revealed a 5' CAP-proximal, strong stem-loop presenting an element similar to multiple start-site element downstream-1 (GCTCGG) in the loop region. RNA-EMSAs performed with the labeled RI alpha 1b 5' UTR showed stabilization of a protein/RNA complex in extracts from 8-(4-chlorophenyl)thio-cAMP stimulated Sertoli cells. This complex was abolished by mutation of the multiple start-site element downstream-1-like element. Our findings indicate that there is a cAMP-mediated induction of RI alpha expression at the post-transcriptional level, dependent on the 5' UTR of RI alpha 1b mRNA.

By using 5' RACE on rat testis cDNA we identified three alternatively spliced mRNAs of the RIalpha subunit of cAMP-dependent protein kinase that differed in their 5' untranslated regions. Two of these 5'-regions showed similarity with the human RIalpha exons 1a and 1b, while the third (1c) constituted a novel mRNA splice variant. Northern blot analysis showed that the 1c mRNA was specifically expressed in testis and only in postmeiotic germ cells. In contrast, the RIalpha 1b and RIalpha 1a mRNAs were present both in premeiotic germ cells and somatic cells of the testis, and the expression of both RIalpha 1a and 1b mRNAs were stimulated by cAMP in Sertoli cells. In sperm, the RIalpha protein was expressed after meiosis, and targeted to various subcellular structures via anchoring proteins. The RIalpha 1c haploid-specific mRNA, therefore, may be important for the regulation of RIalpha expression in sperm.

The C/EBP (CCAAT/enhancer-binding protein) family of transcription factors is important for differentiation, lipid biosynthesis, and metabolism. Here, we demonstrate for the first time the presence of C/EBP alpha, beta, delta, and zeta messenger RNA (mRNA) and protein in Sertoli cell primary cultures. Treatment with FSH or 8-CPTcAMP strongly induced C/EBP beta mRNA above basal levels with rapid and transient kinetics in Sertoli cell primary cultures as well as in whole testes from hypophysectomized rats. Whereas C/EBP beta mRNA was induced approximately 50-fold, C/EBP delta mRNA was induced 5- to 8-fold by cAMP in Sertoli cells. Messenger RNA for C/EBP beta and delta were induced by inhibition of protein synthesis with cycloheximide and cycloheximide acted synergistically with cAMP. Immunoblots with C/EBP antibodies demonstrated a strong induction of C/EBP beta, delta, and zeta by cAMP. Electrophoretic mobility shift analysis of nuclear proteins from cAMP-treated Sertoli cells using a C/EBP consensus oligonucleotide and antibodies revealed specific binding of C/EBP/DNA complexes, the majority of which were supershifted by C/EBP beta antibody. Transfections of Sertoli cells with a C/EBP reporter construct showed approximately 3-fold induction of reporter gene activity by cAMP. In contrast, the reporter gene vector with a mutated form of the C/EBP binding site, was almost unresponsive to cAMP in transfections of Sertoli cells. Furthermore, C/EBP beta expression increased the activities of two promoters known to be cAMP-responsive in Sertoli cells. Thus, the early induction of C/EBP isoforms by cAMP may play a role in FSH-dependent regulation of late response genes in Sertoli cells.