Prognostic biomarkers for immunotherapy with ipilimumab in metastatic melanoma.

New therapies, including the anti-cytotoxic T lymphocyte antigen (CTLA)-4 antibody, ipilimumab, is approved for metastatic melanoma. Prognostic biomarkers need to be identified, because the treatment has serious side effects. Serum samples were obtained before and during treatment from 56 patients with metastatic or unresectable malignant melanoma, receiving treatment with ipilimumab in a national Phase IV study (NCT0268196). Expression of a panel of 17 inflammatory-related markers reflecting different pathways including extracellular matrix remodeling and fibrosis, vascular inflammation and monocyte/macrophage activation were measured at baseline and the second and/or third course of treatment with ipilimumab. Six candidate proteins [endostatin, osteoprotegerin (OPG), C-reactive protein (CRP), pulmonary and activation-regulated chemokine (PARC), growth differentiation factor 15 (GDF15) and galectin-3 binding-protein (Gal3BP)] were persistently higher in non-survivors. In particular, high Gal3BP and endostatin levels were also independently associated with poor 2-year survival after adjusting for lactate dehydrogenase, M-stage and number of organs affected. A 1 standard deviation increase in endostatin gave 1·74 times [95% confidence interval (CI) = 1·10-2·78, P = 0·019] and for Gal3BP 1·52 times (95% CI = 1·01-2·29, P = 0·047) higher risk of death in the adjusted model. Endostatin and Gal3BP may represent prognostic biomarkers for patients on ipilimumab treatment in metastatic melanoma and should be further evaluated. Owing to the non-placebo design, we could only relate our findings to prognosis during ipilimumab treatment.

Low Expression of miR-424-3p is Highly Correlated with Clinical Failure in Prostate Cancer.

Prostate cancer (PC) is a highly heterogenous disease and one of the leading causes of mortality in developed countries. Recently, studies have shown that expression of immune checkpoint proteins are directly or indirectly repressed by microRNAs (miRs) in many types of cancers. The great advantages of using miRs based therapy is the capacity of these short transcripts to target multiple molecules for the same- or different pathways with synergistic immune inhibition effects. miR-424 has previously been described as a biomarker of poor prognosis in different types of cancers. miR-424 is also found to target both the CTLA-4/CD80- and PD-1/PD-L1 axis. In the present study, the clinical significance of miR-424-3p expression in PC tissue was evaluated. Naïve radical prostatectomy specimens from 535 patients was used for tissue microarray construction. In situ hybridization was used to evaluate the expression of miR-424-3p and immunohistochemistry was used for CTLA-4 protein detection. In univariate- and multivariate analyses, low expression of miR-424-3p was significant associated with clinical failure-free survival, (p = 0.004) and p = 0.018 (HR:0.44, CI95% 0.22-0.87). Low expression of miR-424-3p also associated strongly with aggressive phenotype of PC. This highlight the importance of miR-424-3p as potential target for therapeutic treatment in prostate cancer.

The alpha-subunit mRNAs for Gs and Go2 are differentially regulated by protein kinase A and protein kinase C in rat Sertoli cells.

In the present study, we have examined regulatory effects of protein kinase A and protein kinase C activation by 8-CPTcAMP and TPA, respectively, on mRNAs for various G protein alpha-subunits and corresponding immunoreactive proteins in rat Sertoli cells. Gs alpha and Go alpha mRNA levels were transiently increased 1.5-fold and 4-fold, respectively, by 8-CPTcAMP in cultured Sertoli cells. This up-regulation of mRNAs for Gs alpha and Go alpha was also observed when Sertoli cells were incubated in the presence of FSH. When protein synthesis was inhibited by cycloheximide, the cAMP-mediated stimulation of Gs alpha mRNA was abolished, whereas Go alpha mRNA was superinduced to a 50- to 100-fold higher level than basal. Activation of protein kinase C with TPA had a strong, synergistic effect on cAMP-mediated stimulation of Gs alpha mRNA, whereas the cAMP-mediated stimulation of Go alpha mRNA was completely blocked. Surprisingly, changes in mRNA levels were not accompanied by any alterations in the levels of immunoreactive Gs alpha and Go alpha proteins.

Different mechanisms are involved in cAMP-mediated induction of mRNAs for subunits of cAMP-dependent protein kinases.

The present study addresses possible mechanisms through which cAMP mediates its effects on mRNA levels for the subunits of protein kinase A (PKA) and the cellular protooncogene, c-fos. Messenger RNAs for the PKA subunits (RI alpha, RII alpha, RII beta, and C alpha) were regulated by cAMP with similar kinetics in Sertoli cells. However, effects of cAMP on the PKA mRNAs were slow compared to a well characterized cAMP responsive gene, c-fos. The magnitude of stimulation was dramatically different between the various PKA subunits, in that RII beta mRNA increased more than 50-fold while the mRNAs for the other subunits were induced only two to four times. Separation of nuclear and cytoplasmic RNA demonstrated that mRNAs for PKA subunits were stimulated to the same extent in these two cellular compartments. The more rapid induction of c-fos mRNA by cAMP, compared to the mRNA for RII beta, was also seen at the level of transcription. Maximal transcription rate for c-fos, RI alpha, and C alpha were observed after 30 min, whereas that for RII beta was increasing during the 2-h period examined. Transcriptional activation of the RI alpha gene also appeared faster than that for RII beta. When Sertoli cells were incubated with 8-(4-chlorophenylthio) cAMP and cycloheximide, a potent inhibitor of protein synthesis, we observed a super-induction of the mRNAs for c-fos (10-fold) and RI alpha (2-fold).(ABSTRACT TRUNCATED AT 250 WORDS)

Protein kinase C activation by 12-O-tetradecanoylphorbol 13-acetate modulates messenger ribonucleic acid levels for two of the regulatory subunits of 3',5'-cyclic adenosine monophosphate-dependent protein kinases (RII beta and RI alpha) via multiple and distinct mechanisms.

Messenger RNAs (mRNA) for two of the regulatory subunits of cAMP-dependent protein kinases (PKA), RII beta and RI alpha, are transiently (maximal levels at 6 h) stimulated by 12-O-tetradecanoylphorbol 13-acetate (TPA) in cultured rat Sertoli cells in a time- and concentration-dependent manner. Whereas TPA (10(-7) M) stimulated RII beta mRNA 11 +/- 2.8 fold (mean +/- SEM), mRNA levels for RI alpha increased only 2.5 +/- 0.6-fold (mean +/- SEM). No effects of TPA on the other subunits of PKA (RII alpha, C alpha) were observed. TPA-dependent accumulation of mRNAs for RII beta and RI alpha was observed to the same extent in nucleus and cytoplasm. We have previously shown that mRNA levels for all the PKA subunits are increased by cAMP, particularly that of RII beta (greater than 50-fold). TPA modulated the stimulatory effects of cAMP on RII beta and RI alpha mRNAs in opposite directions. Whereas treatment with both 8-CPTcAMP and TPA gave an additive effect on RI alpha mRNA, TPA reduced the cAMP-dependent increase in RII beta mRNA. Although the mRNA for RII beta had returned to basal levels after 24 h of incubation with TPA, the presence of TPA still inhibited cAMP-dependent induction of mRNA for RII beta. In contrast, similar TPA treatment did not influence the subsequent cAMP-dependent stimulation of RI alpha mRNA. Preincubation with 8-CPTcAMP did not influence TPA-dependent stimulation of mRNAs for either RII beta or RI alpha. TPA induction of RII beta mRNA was completely blocked by cycloheximide (an inhibitor of protein synthesis), whereas that of RI alpha was not. The inhibitory effect of TPA on cAMP stimulation of RII beta mRNA was independent of ongoing protein synthesis. These results indicate that TPA induction of mRNAs for RI alpha and RII beta involves multiple and distinct mechanisms. The stimulatory effect of TPA on RI alpha mRNA levels and the inhibitory effect of TPA on cAMP-stimulated RII beta mRNA expression are probably mediated through stable factors, whereas proteins with rapid turnover or factors induced by TPA are involved in the stimulatory effect of TPA on RII beta mRNA.

Adenosine 3',5'-monophosphate-dependent stabilization of messenger ribonucleic acids (mRNAs) for protein kinase-A (PKA) subunits in rat Sertoli cells: rapid degradation of mRNAs for PKA subunits is dependent on ongoing RNA and protein synthesis.

cAMP treatment of primary cultures of Sertoli cells is associated with a transient stimulatory effect on mRNA levels for various protein kinase-A (PKA) subunits. We have previously shown that the induction of mRNA for regulatory subunit II beta (RII beta) is due at least partly to transcriptional activation. In the present study we investigate possible regulatory effects of (Bu)2cAMP on the degradation of mRNAs for various PKA subunits in rat Sertoli cells. We demonstrate subunit specific differences in the decay of mRNAs for the various PKA subunits. When (Bu)2cAMP was removed from Sertoli cell cultures after 6 h of stimulation, there was a rapid decay of mRNAs for both RII beta and RI alpha (half-lives, approximately 3 h). In contrast, mRNA levels for RII alpha continued to increase. Removal of (Bu)2cAMP after a longer period of treatment revealed a similar decay of mRNAs for all of the PKA subunits, with half-lives of approximately 3 h. Incubation of Sertoli cells for 12 h with (Bu)2cAMP, followed by continued incubation in the absence and presence of (Bu)2cAMP as well as in the presence of actinomycin-D (an inhibitor of RNA synthesis), revealed (Bu)2cAMP mediated stabilization of mRNA for the RII beta subunit. Interestingly, actinomycin-D as such stabilized mRNAs for all PKA subunits. Similar treatment with cycloheximide (an inhibitor of protein synthesis) revealed distinct differences between the RI alpha and C alpha subunits vs. the RII subunits; cycloheximide reduced the decay of both RII beta and RII alpha mRNAs, whereas steady state levels of mRNAs for RI alpha and C alpha actually increased after cycloheximide treatment of previously (Bu)2cAMP-stimulated cultures. Cycloheximide treatment also increased basal levels of mRNAs for RI alpha and C alpha, whereas basal levels of RII beta and RII alpha mRNAs were not influenced. These studies indicate that the degradation of mRNAs for the various PKA subunits is subject to different regulation by (Bu)2cAMP, and that ongoing RNA and protein synthesis is required for rapid degradation of all PKA subunits.

Isoform-specific regulation of the CCAAT/enhancer-binding protein family of transcription factors by 3',5'-cyclic adenosine monophosphate in Sertoli cells.

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.

Characterization of the 5'-flanking region of the gene for the cAMP-inducible protein kinase A subunit, RIIbeta, in Sertoli cells.

Activation of cyclic AMP-dependent protein kinases (protein kinase A, PKA) by gonadotropins and cyclic AMP (cAMP) plays an important role in the regulation of testicular functions. A regulatory subunit, RIIbeta, of PKA is transcriptionally induced in rat Sertoli cells in response to treatment with cAMP. The present study addresses regulatory mechanisms leading to increased transcription of the rat RIIbeta gene. We have localized a footprint which overlaps one of the major transcription initiation sites in the basal promoter (-293 to -123). One of the proteins binding this sequence belongs to the NF-1 family of transcription factors. We also observed binding to a basic helix-loop-helix (bHLH) response element. Furthermore, transfection studies of various 5'-deletions of the rat RIIbeta gene in primary cultures of rat Sertoli cells and in peritubular cells revealed the presence of an upstream region (-723 to -395, cAMP-responsive region) inhibiting basal expression from the rat RIIbeta gene only in Sertoli cells. This region was found to enhance cAMP responsiveness in Sertoli cells but not in peritubular cells. Interactions with downstream elements seemed to be important for the function of the cAMP-responsive region. Although some short stretches reveal homology to the cAMP-responsive regions of other slowly cAMP-responding genes, and an AP-1-like element is present, no strong resemblance to any known regulatory element responsive to cAMP is found.

Method of transfection affects the cAMP-mediated induction of the RIIbeta subunit of protein kinase A in Sertoli cells: inhibition of response by increase in intracellullar calcium.

mRNA for the regulatory subunit RIIbeta of cAMP-dependent protein kinase is stimulated more than 50-fold by cAMP in primary cultures of rat Sertoli cells. We have previously shown that this induction involves regulation of transcriptional activation as well as mRNA stabilization. The rat RIIbeta gene contains no cAMP response element (CRE), and the induction of RIIbeta mRNA is slow and requires on-going protein synthesis. When a construct containing the 5'-flanking region of the RIIbeta gene upstream of a CAT reporter was transfected into Sertoli cells by the calcium phosphate method, low and variable responses to cAMP (three- to fivefold) were observed, whereas a 15- to 20-fold increase in reporter activity by cAMP was observed after lipofectamine transfection. Interestingly, when a vector containing CRE elements upstream of a reporter gene was transfected into Sertoli cells, the responses to cAMP were similar regardless of the transfection method used. We have also demonstrated that increased intracellular levels of calcium by A23187 and thapsigargin dramatically inhibit cAMP-mediated induction of RIIbeta mRNA, but not the mRNA for the CRE-containing RIalpha gene. Furthermore, decreased cAMP responsiveness of endogenous RIIbetamRNA (but not RIalpha) was also observed in calcium phosphate-transfected Sertoli cells but not in lipofectamine-transfected cells. Thus, calcium-mediated reduction in cAMP response appears to be a gene-specific phenomenon.

Expression of serotonin receptors 2B and 4 in human prostate cancer tissue and effects of their antagonists on prostate cancer cell lines.

OBJECTIVES: Overexpression of receptors to neuroendocrine (NE) cell products has been suggested to contribute to development of hormone-refractory prostate cancer (HRPC). In this study, we evaluated the expression of 5-HTR2B and 5-HTR4 in HRPC, and the effects of their antagonist on PC cell line growth. METHODS: Proteins and mRNA expression was determined by immunohistochemistry, western blot and RT-PCR. Growth inhibition of PC cell lines was determined in vitro using ELISA-BrdU proliferation assay and cell cycle was evaluated by flow cytometry. RESULTS: Immunostaining of 5-HTR2B was observed in low-grade and high-grade tumours, PIN and BPH cells, and in vascular endothelial cells, whereas 5-HTR4 was found predominantly in high-grade tumours. This result was confirmed by western blot analysis. At the mRNA level, 5-HTR4 mRNA was expressed in DU145 and LNCaP cells. Antagonists to both receptor subtypes inhibited proliferation of PC cells in a dose-dependent manner. CONCLUSIONS: The present result indicate that 5-HTRs are present at various tumour stages and that antagonists to these receptors can inhibit the proliferative activity of androgen-independent PC cell lines.

Protein kinase C activation and positive and negative agonist regulation of 3',5'-cyclic adenosine monophosphate levels in cultured rat Sertoli cells.

The present study examines the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on agonist-regulated 3',5'-cyclic adenosine monophosphate (cAMP) formation and cAMP-mediated effects in cultured Sertoli cells from immature rats. Concentration-dependent stimulation of cAMP levels by follicle-stimulating hormone (FSH) was inhibited dramatically by the coaddition of 100 nmol/l TPA, which exerted a similar inhibition of glucagon- and isoproterenol-stimulated cAMP production. These results show that protein kinase C (PKC) activation by TPA attenuates Gs-protein-mediated agonist activation of cAMP production. (-)-N6(R)-Phenylisopropyladenosine (L-PIA), an A1-adenosine receptor agonist, inhibited cAMP stimulation by FSH in a concentration-dependent manner. When L-PIA was added in increasing concentrations simultaneously with 100 nmol/l TPA, the L-PIA still inhibited FSH-stimulated cAMP production in a concentration-dependent manner. In the presence of TPA, the half-inhibitory concentration (IC50) for L-PIA inhibition of cAMP formation was reduced by more than one order of magnitude, indicating that PKC activation by TPA increases the sensitivity of Sertoli cells to Gi-protein-mediated agonist inhibition of cAMP production. The inhibitory effects of TPA on FSH-stimulated cAMP production were still observed when cAMP phosphodiesterase activity was inhibited by 1 mmol/l methylisobutylxanthine or when the activity of G alpha i-protein was eliminated by pretreatment with 100 micrograms/l pertussis toxin. Taken together, the results indicate that PKC activation inhibits agonist-dependent stimulation of cAMP production by phosphorylation of components common to all the activating agonists used, and not via stimulation of G(i)-protein activity or degradation of cAMP by cAMP phosphodiesterase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibitors of RNA and protein synthesis stabilize messenger RNA for the RII beta subunit of protein kinase A in different cellular compartments.

Messenger RNA for RII beta is transiently induced (greater than 50-fold) by cAMP analogs in primary cultures of rat Sertoli cells. The induction is dependent on protein synthesis. We have previously shown that mRNA for RII beta is stabilized by cAMP, as well as inhibitors of transcription and translation. This indicated that rapid degradation of RII beta mRNA involved a protein with a rapid turnover and its corresponding mRNA. The two RNA synthesis inhibitors used in the present study stabilized both nuclear and cytoplasmic RII beta mRNA, whereas inhibition of protein synthesis stabilized RII beta mRNA in the cytoplasm only. These results indicate that only cytoplasmic degradation of RII beta mRNA is dependent on a protein with high turnover. In contrast, nuclear degradation appears to be dependent on an RNA with a short half-life, not involving protein synthesis.

Half-lives of different sized mRNAs for the PKA subunit RI alpha are regulated differently in response to inhibition of transcription and translation.

The RI alpha mRNA level is induced 3-5 times by FSH or cAMP analogs in primary cultures of rat Sertoli cells. In rat tissues, the RI alpha gene gives rise to three different mRNAs of different size: 3.2, 2.9 and 1.7 kb. In the present study we report that the 1.7 kb transcript has a shorter half-life than the two other mRNAs. In cells which had been pre-stimulated with a cAMP analog, inhibition of transcription stabilizes the two larger, but not the smaller sized RI alpha mRNA. However, in contrast, inhibition of protein synthesis stabilizes all the RI alpha mRNAs. Thus, degradation of various mRNAs coding for the same protein reveals different dependencies on transcription and translation.

Regulation of protein kinase A subunits by cyclic adenosine 3',5'-monophosphate in a mouse Sertoli cell line (MSC-1): induction of RII beta messenger ribonucleic acid is independent of continuous protein synthesis.

We report the basal and cAMP-regulated expression of protein kinase A (PKA) subunits in a mouse Sertoli cell line (MSC-1). Of the PKA subunits expressed by these cells (RI alpha, RII alpha, RII beta, C alpha, C beta), only RII beta was regulated by cAMP. An approximately 8-fold induction of RII beta mRNA and a 3-fold induction of RII beta protein was observed during 48 h of cAMP-stimulation. This cAMP-mediated RII beta mRNA induction, reaching maximal levels after approximately 12 h, did not require ongoing protein synthesis. Fairly rapid decay of maximally induced RII beta mRNA was observed after removal of cAMP (t1/2 approximately 5 h). Further, ongoing transcription and translation were necessary for rapid degradation of RII beta mRNA. Thus, the MSC-1 cells expressed all the PKA subunits present in primary cultures of Sertoli cells and responded to cAMP with increased levels of RII beta at both mRNA and protein levels. Although the nature of some of these responses distinguished the observations in MSC-1 cells from previously described responses in primary cultures, these cells may prove to be useful in future studies addressing cAMP-mediated gene regulation.

Calcium/phospholipid-dependent protein kinases in rat Sertoli cells: regulation of androgen receptor messenger ribonucleic acid.

The possibility that Sertoli cell responses to testosterone are modulated by the calcium/phospholipid-dependent protein kinase (protein kinase C; PKC) was examined in rat Sertoli cells in culture. Both soluble and particulate cell fractions showed low constitutive phosphotransferase activity. Incubation with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA; 10(-7) M) was associated with a transient induction in both cell fractions of calcium/phosphatidylserine-dependent PKC activity, which was elevated from 15 min to 1 h. Consistent with this, mRNAs for the calcium/phospholipid-dependent isomeric forms of PKC (alpha, beta, and gamma) were detected. The expression levels of mRNAs for PKCalpha and PKCbeta were also up-regulated (2.5- to 3-fold) by TPA (10(-7) M), but these effects were much slower (peaking after 12 h) than those on phosphotransferase activity. In the presence of TPA (10(-7) M), expression of androgen receptor (AR) mRNA showed a transient time-dependent down-regulation ( approximately 70%), in which the nadir was reached after 6 h and baseline expression was again obtained after 12 h. The regulatory effect of PKC activation on AR mRNA was confirmed by the absence of response to a biologically inactive phorbol ester. A concentration-dependent decrease (half-maximal effect at approximately 10(-8) M TPA) of AR mRNA was also observed. These data suggest that Sertoli cell responses to testosterone may be inhibited by a transiently active PKC with a wide intracellular distribution.

Phosphodiesterase 4D and protein kinase a type II constitute a signaling unit in the centrosomal area.

The mediation of cAMP effects by specific pools of protein kinase A (PKA) targeted to distinct subcellular domains raises the question of how inactivation of the cAMP signal is achieved locally and whether similar targeting of phosphodiesterases (PDEs) to sites of cAMP/PKA action could be observed. Here, we demonstrate that Sertoli cells of the testis contain an insoluble PDE4D3 isoform, which is shown by immunofluorescence to target to centrosomes. Staining of PDE4D and PKA shows co-localization of PDE4D with PKA-RIIalpha and RIIbeta in the centrosomal region. Co-precipitation of RII subunits and PDE4D3 from cytoskeletal extracts indicates a physical association of the two proteins. Distribution of PDE4D overlaps with that of the centrosomal PKA-anchoring protein, AKAP450, and AKAP450, PDE4D3, and PKA-RIIalpha co-immunoprecipitate. Finally, both PDE4D3 and PKA co-precipitate with a soluble fragment of AKAP450 encompassing amino acids 1710 to 2872 when co-expressed in 293T cells. Thus, a centrosomal complex that includes PDE4D and PKA constitutes a novel signaling unit that may provide accurate spatio-temporal modulation of cAMP signals.

Cyclic AMP regulates expression of the RI alpha subunit of cAMP-dependent protein kinase through an alternatively spliced 5' UTR.

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.

Novel alternatively spliced mRNA (1c) of the protein kinase A RIα subunit is implicated in haploid germ cell specific expression.

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.

Reciprocal regulation of mRNA and protein for subunits of cAMP-dependent protein kinase (RI alpha and C alpha) by cAMP in a neoplastic B cell line (Reh).

The present study examines the activity, levels of expression and regulation of cAMP-dependent protein kinase subunits during cAMP-mediated inhibition of Reh cell proliferation. Human Reh cells express mRNAs for the RI alpha and C alpha subunits of cAK at high levels and are practically devoid of cAMP-dependent protein kinase type II. Treatment with isoproterenol, forskolin, or a cAMP analog increased RI alpha mRNA in a time- and concentration-dependent manner (maximal, 4-fold, at 4-8 h). Messenger RNA for C alpha was also stimulated by cAMP, although with slower kinetics (maximal, 2-fold, at 16-24 h). Nuclear run-on assays showed a 2-fold increase in RI alpha gene transcription, whereas that of C alpha was unchanged. In spite of the stimulatory effects of cAMP on mRNAs for both RI alpha and C alpha, phosphotransferase activity and specific [3H]cAMP binding decreased rapidly after treatment with either cAMP or forskolin. Interestingly, the decrease in R and C activity preceded the increase in RI alpha and C alpha mRNA levels, raising the question whether increased mRNA levels may be secondary to the decrease in RI alpha or C alpha protein. The finding that the protein synthesis inhibitor cycloheximide gave changes in RI alpha and C alpha mRNA similar to cAMP and that co-treatment with cycloheximide and cAMP resulted in additive effects tend to support this notion.

Regulation of tissue inhibitor of metalloproteinases-1 in rat Sertoli cells: induction by germ cell residual bodies, interleukin-1alpha, and second messengers.

In the testis, FSH has been shown to induce the expression and secretion of tissue inhibitor of metalloproteinases-1 (TIMP-1) from Sertoli cells in vitro. This study was performed to elucidate further the cellular origin of testicular TIMP-1 and its expression by hormonal and paracrine factors. This is the first report on the expression of testicular TIMP-1 in vivo. TIMP-1 mRNA in whole testis was decreased after hypophysectomy and strongly increased by the injection of FSH-S17 to hypophysectomized rats. Primary cultures of both peritubular and Sertoli cells showed basal expression of TIMP-1 mRNA. In contrast, we were unable to detect TIMP-1 mRNA in Leydig cells, freshly isolated immature germ cells (primary spermatocytes and spermatids), or residual bodies. We further show that treatment of Sertoli cells with 8-(4-chlorophenyl)thio-cAMP (8-CPTcAMP) in combination with 12-O-tetradecanoylphorbol 13-acetate (TPA) or Ca(2+) inducers (calcium ionophore A23187 or thapsigargin) had additive (TPA) and synergistic effects (Ca(2+)) on the level of TIMP-1 mRNA and secreted protein. We also show that both the level of TIMP-1 mRNA and secreted protein from Sertoli cells were strongly increased by residual bodies, as well as by the cytokine interleukin-1alpha. TIMP-1 was not up-regulated by either 8-CPTcAMP or interleukin-1alpha in peritubular cells. In contrast to the regulated secretory fraction of TIMP-1, we also detected constitutively expressed immunoreactive TIMP-1 in the nucleus of Sertoli cells, suggesting a role of nuclear TIMP-1 in these cells. In conclusion, our data show that secretion of TIMP-1 from Sertoli cells is highly regulated by hormonal and local processes in the testis, indicating that TIMP-1 is of physiological importance during both testicular development and spermatogenesis.