To address a possible role of type 1 and 2A serine/threonine protein phosphatases (PP1 and PP2A) in regulating granulosa cell hormonal responses, we investigated the effects of okadaic acid (OA) on FSH- and cAMP-induced steroidogenesis in these cells. When added alone (0.01-1 nmol/l), the cell-permeant phosphatase inhibitor did not affect progesterone and 3 beta-hydroxysteroid dehydrogenase/delta 5-4 isomerase (3 beta-HSD) enzyme activity, whereas when added with FSH it dose-dependently augmented (minimal effective dose, 0.1 nmol/l) gonadotropin-stimulated steroidogenesis in cultured granulosa cells. A similar stimulatory effect of the toxin was observed in cells cultured for 48 h with the cell-permeant analogue dibutyryl cAMP (1 mmol/l), or when granulosa cells were stimulated with the cAMP-inducing agents cholera toxin (1 microgram/ml), forskolin (15 mumol/l) or 1-methyl-3-isobutyl-xanthine (0.1 mmol/l). The observed effect of OA on FSH-supported granulosa cell steroidogenesis was not a consequence of increased cAMP generation, and time course experiments also revealed that a minimal time period of 12 h was necessary for OA (0.1 and 1 nmol/l) to significantly enhance FSH-induced progesterone and 3 beta-HSD enzyme activity. Since OA also inhibits the dephosphorylation of protein kinase C (PKC) substrates, we also compared the effect of OA and the PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA) on FSH-induced granulosa cell steroidogenic activity. While activation of the PKC pathway with the tumor promoter TPA (10 nmol/l) inhibited progesterone and cAMP accumulation in FSH-stimulated granulosa cells, treatment with OA augmented steroidogenesis and did not affect gonadotropin-induced cAMP generation. Collectively these results suggest that PP1 and PP2A may be important in regulating the phosphorylation state of proteins implicated in the cAMP-protein kinase A-stimulated steroidogenic activity of these cells.

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Purpose: Ubiquitin proteasome pathway (UPP) failure has been reported in case of rabbit Limbal Stem Cell Deficiency (LSCD). In the present study, we examined the effect of LSCD on autophagy in comparison to LSCD's effect on UPP. Methods: Rabbits with surgically induced LSCD were used. Corneal epithelial cells (CEC) were collected to measure the levels of autophagy and UPP biomarkers. Rabbit oral epithelial cells were isolated, cultured and treated with autophagy and proteasome inhibitors. Results: Immunofluorescent staining showed that both pathways were positive in normal corneal epithelium. However, while proteasome subunits were decreased in LSCD-CEC, autophagy biomarker MAPLC3B and ATG12-ATG5 complex were significantly increased in LSCD-CEC, compared to healthy CEC. These results indicate that LSCD-induced impairment of UPP may cause a compensatory stimulation of autophagy. However, despite autophagy up regulation, damaged and unwanted proteins, such as modified keratin K4 and K13 aggregates, still deposited and accumulated in LSCD-CEC without clearance, possibly contributing to CEC haziness. Proteasome inhibition in cultured oral epithelial cell sheet caused an increase in the expression of MAPLC3B and ATG12-ATG5 complex, supporting the observation that when UPP is failing, autophagy is stimulated. When autophagy was inhibited with chloroquine, proteasome activity was significantly increased compared to untreated cells. In addition, there a slight decrease in unmodified K4 and K13 expression levels with no keratin high molecular weight deposition when autophagy was inhibited. Conclusions: We report that inhibition of autophagy leads to an activation in proteasome activity, which may stimulate protein degradation to alleviate the symptoms of LSCD.