Cyclic AMP inhibits agonist-induced heparin-binding EGF gene expression independently of effects on p42/p44 MAPK activation

Heparin-binding EGF-like growth factor (HB-EGF) mRNA levels are increased up to 20-fold in RIE-1 cells by two agonists that act through distinct receptor types. We demonstrated a common requirement for p42/p44 mitogen-activated protein kinase (MAPK) in this response using the selective MAPK kinase (MEK) inhibitor, PD 098059. Agonist-mediated induction of HB-EGF mRNA was markedly suppressed in cells that had been treated with cyclic AMP-elevating agents. In contrast, the activation of p42 MAPK in response to agonists was not affected by raising cellular cyclic AMP levels. We conclude that cyclic AMP negatively regulates the HB-EGF gene, but that the inhibitory action is either independent of the p42/p44 MAPK pathway or the site of action is distal to MAPK activation.     

Vasoactive intestinal peptide suppresses ovarian granulosa cell apoptosis in vitro by up-regulating Bcl-2 gene expression

Vasoactive intestinal peptide (VIP) is an endogenous peptide showing a rich profile of biological activities. Within ovaries, VIP directly regulates the ovarian functions, including granulosa cells (GCs) development. In the present study, the effects of VIP on proliferation and apoptosis in goose granulosa cells were demonstrated and its underlying mechanism investigated. A strategy of RNAi-mediated "gene silencing" of Bcl-2 (RV-Bcl-2), over-expression of Bcl-2 (JLV-Bcl-2) synthesis, and exogenous VIP was used to treat goose GCs. The results showed the amounts of Bcl-2 protein were negatively correlated with apoptosis of goose GCs in all experimental groups. Compared with other control groups, apoptosis was decreased in goose GCs following treatment of 100 nM VIP, and the amount of Bcl-2 protein was increased (P < 0.05) increased. However, VIP failed to exert an effect on cell proliferation (P > 0.05). In conclusion, the exogenous VIP plays an important role in inhibiting apoptosis of goose GCs via inducing Bcl-2 gene expression.     

Gap junction communication and connexin 43 gene expression in a rat granulosa cell line: regulation by follicle-stimulating hormone

Follicle-stimulating hormone is the major regulator of growth and development of antral follicles in the ovary. Granulosa cells (GCs) in these follicles are coupled via gap junctions (GJs) consisting of connexin 43 (Cx 43). Because we and others have found that Cx 43 and GJs, respectively, are more abundant in large antral follicles compared with small antral and preantral follicles, we hypothesized that FSH may control Cx 43 gene expression, GJ formation, and intercellular communication. To directly address these points, we chose a rat GC line (GFSHR-17) expressing the FSH receptor and the Cx 43 gene. The functionality of FSH receptors was shown by the effects of porcine FSH, namely cell rounding, reduced cellular proliferation, and stimulation of progesterone production of GFSHR-17 cells, which are effects that were detectable within hours. Treatment with FSH also statistically significantly increased Cx 43 mRNA levels, as shown after 6 to 9 h in Northern blots. These effects were antedated by altered GJ communication, which was observed within seconds. Using a single-cell/whole-cell patch clamp technique, we showed that FSH rapidly and reversibly enhanced electrical cell coupling of GFSHR-17 cells. Increased GJ communication was associated with statistically significantly decreased phosphorylation of Cx 43, which was observed within 10 min after FSH addition, during immunoprecipitation experiments. Our results demonstrate, to our knowledge for the first time, that the gonadotropin FSH acutely and directly stimulates intercellular communication of GFSHR-17 cells through existing GJs. Moreover, FSH also increases levels of Cx 43 mRNA. These changes are associated with reduced proliferation and enhanced differentiation of GFSHR-17 cells. In vivo factors in addition to FSH may be involved in the regulation of GJ/GJ communication between GCs in the follicle, but our results suggest that improved cell-to-cell coupling, enhanced Cx 43 gene expression, and possibly, formation of new GJs are direct consequences of FSH receptor activation and may antedate and/or initiate the pivotal effects of FSH on GCs.     

Morphine withdrawal inhibits IL-12 induction in a macrophage cell line through a mechanism that involves cAMP

There are very few studies that examine the effects that morphine withdrawal has on immune functioning, and of these even fewer describe the mechanisms by which withdrawal brings about these changes. Our previous work demonstrated that morphine withdrawal contributed to Th cell differentiation by biasing cells toward the Th2 lineage. A major finding from these studies was that IL-12 was decreased following withdrawal, and it was concluded that this decrease may be a mechanism by which morphine withdrawal is mediating Th2 polarization. Therefore, it was the aim of the current studies to develop an in vitro model to examine the process of morphine withdrawal and to understand the signaling mechanisms that withdrawal may use to effect IL-12 production through the use of this model. It was demonstrated and concluded that morphine withdrawal may be effecting IL-12 production by increasing cAMP levels, which activates protein kinase A. Protein kinase A activation then prevents the phosphorylation and subsequent degradation of IkappaB, which in turn prevents translocation of the NF-kappaB p65 subunit to the nucleus to transactivate the IL-12 p40 gene, ultimately resulting in decreased IL-12 production following LPS stimulation.     

Enhanced cell aggregation in Dictyostelium discoideum by ATP activation of Cyclic AMP receptors.

Cell aggregation in the cellular slime mold Dictyostelium discoideum is mediated by cyclic AMP. In the presence of ATP the onset of cell aggregation is enhanced and cyclic AMP receptors are activated. The number of phosphorylation sites is species dependent, and two main phosphorylated proteins with MW of, respectively, 20,000 and 15,000 are localized.     

MiR-92a inhibits porcine ovarian granulosa cell apoptosis by targeting Smad7 gene

Smad7 has a key role in apoptosis of mammalian ovarian granulosa cells (GCs), as it antagonizes and fine-tunes transforming growth factor β (TGFβ) signaling. This study demonstrates that miR-92a regulates GC apoptosis in pig ovaries by targeting Smad7 directly. The expression level of miR-92a was down-regulated in atretic porcine follicles, whereas miR-92a expression led to inhibition of GC apoptosis. The Smad7 gene was identified as a direct target of miR-92a using a dual-luciferase reporter assay. Transfection of GCs with miR-92a mimics decreased Smad7 mRNA and protein levels, whereas expression of an miR-92a inhibitor in GCs had the opposite effect. In addition, knockdown of Smad7 prevented GC apoptosis in cells that expressed the miR-92a inhibitor.     

Targeting a newly established spontaneous feline fibrosarcoma cell line by gene transfer

Fibrosarcoma is a deadly disease in cats and is significantly more often located at classical vaccine injections sites. More rare forms of spontaneous non-vaccination site (NSV) fibrosarcomas have been described and have been found associated to genetic alterations. Purpose of this study was to compare the efficacy of adenoviral gene transfer in NVS fibrosarcoma. We isolated and characterized a NVS fibrosarcoma cell line (Cocca-6A) from a spontaneous fibrosarcoma that occurred in a domestic calico cat. The feline cells were karyotyped and their chromosome number was counted using a Giemsa staining. Adenoviral gene transfer was verified by western blot analysis. Flow cytometry assay and Annexin-V were used to study cell-cycle changes and cell death of transduced cells. Cocca-6A fibrosarcoma cells were morphologically and cytogenetically characterized. Giemsa block staining of metaphase spreads of the Cocca-6A cells showed deletion of one of the E1 chromosomes, where feline p53 maps. Semi-quantitative PCR demonstrated reduction of p53 genomic DNA in the Cocca-6A cells. Adenoviral gene transfer determined a remarkable effect on the viability and growth of the Cocca-6A cells following single transduction with adenoviruses carrying Mda-7/IL-24 or IFN-γ or various combination of RB/p105, Ras-DN, IFN-γ, and Mda-7 gene transfer. Therapy for feline fibrosarcomas is often insufficient for long lasting tumor eradication. More gene transfer studies should be conducted in order to understand if these viral vectors could be applicable regardless the origin (spontaneous vs. vaccine induced) of feline fibrosarcomas.     

Cyclic AMP activation of a triglyceride lipase in broken cell preparations of rat heart

The effect of CAM [cyclic AMP, Mg-ATP, and 3-isobutyl, 1-methylxanthine (MIX)] on triacylglycerol (TG) lipase activity in extracts from heparin-perfused rat heart was determined. TG lipase activity in homogenate, 10,000g supernatant, 105,000g supernatant, ammonium sulfate supernatant, and the eluate from heparin-Sepharose was increased between 62 and 151% when incubated with a combination of 0.3 mM cyclic AMP, 5 mM MgCl2, and 2 mM ATP. The addition of Mg-ATP + cyclic AMP caused a greater activation of TG lipase in the various fractions than did Mg-ATP + MIX or cyclic AMP + MIX. These results suggest that activation may be mediated by the classical cyclic AMP-protein kinase cascade. Control and CAM-stimulated activities were increased by heparin and inhibited by NaCl and protamine sulfate. In the absence of serum in the assay, the CAM system caused a relatively greater stimulation of lipolytic activity in each fraction compared to when serum was present in the assay. However, the absolute values were 6.1 to 16.3-fold greater with serum in the assay than without serum. In a similar manner, TG lipase activity was stimulated by CAM between 1.75 and 4.26-fold at pH 7.4, and only between 1.62 and 2.51-fold at pH 8.1. However, the absolute values at pH 8.1 were 6.77 to 31.83-fold greater than those seen at pH 7.4. These data demonstrate, for the first time, the cyclic AMP activation of a TG lipase above basal levels in cell-free fractions of rat heart. It is intriguing to speculate that the intracellular fraction of lipoprotein lipase may play a role in the hormonal regulation of cardiac TG lipolysis.     

Novel mechanism of cyclic AMP mediated extracellular signal regulated kinase activation in an intestinal cell line

The extracellular signal regulated kinase (ERK1/2) signaling cascade has been implicated as both a pro-apoptotic and anti-apoptotic pathway depending on cell type and context. In the T84 intestinal epithelial cell line, cAMP activates ERK1/2 resulting in the inhibition of apoptosis. Cyclic-AMP signaling relies on the binding and activation of a cAMP binding protein. In most cell types, the majority of this signaling occurs through an isoform of protein kinase A (PKAI or PKAII). Despite evidence to the contrary, we hypothesized that ERK1/2 activation is through a PKA isoform. Pharmacological activators and inhibitors of PKA as well as siRNA were used to further interrogate this potential signaling pathway. Our results demonstrate that at doses sufficient to increase PKA activity, PKAII specific cAMP analogs activate ERK1/2 while PKAI analogs do not. Pharmacological inhibition of the PKAII regulatory subunit and catalytic subunit as well as siRNA knockdown of the catalytic subunit blocks ERK1/2 activation. We conclude that in the T84 cell line, cAMP binding to the PKAII regulatory subunit leads to the subsequent phosphorylation of ERK1/2 and provides insight into the mechanism of cAMP mediated survival signaling in the intestinal epithelium. These results directly implicate PKAII as a mediator of cell survival in T84 cells and provide evidence for an additional means by which cAMP can influence intestinal cell turnover.     

Molecular mechanism of fibronectin gene activation by cyclic stretch in vascular smooth muscle cells

Fibronectin plays an important role in vascular remodeling. A functional interaction between mechanical stimuli and locally produced vasoactive agents is suggested to be crucial for vascular remodeling. We examined the effect of mechanical stretch on fibronectin gene expression in vascular smooth muscle cells and the role of vascular angiotensin II in the regulation of the fibronectin gene in response to stretch. Cyclic stretch induced an increase in vascular fibronectin mRNA levels that was inhibited by actinomycin D and CV11974, an angiotensin II type 1 receptor antagonist; cycloheximide and PD123319, an angiotensin II type 2 receptor antagonist, did not affect the induction. In transfection experiments, fibronectin promoter activity was stimulated by stretch and inhibited by CV11974 but not by PD123319. DNA-protein binding experiments revealed that cyclic stretch enhanced nuclear binding to the AP-1 site, which was partially supershifted by antibody to c-Jun. Site-directed mutation of the AP-1 site significantly decreased the cyclic stretch-mediated activation of fibronectin promoter. Furthermore, antisense c-jun oligonucleotides decreased the stretch-induced stimulation of the fibronectin promoter activity and the mRNA expression. These results suggest that cyclic stretch stimulates vascular fibronectin gene expression mainly via the activation of AP-1 through the angiotensin II type 1 receptor.