JNK and p38 are activated by erythropoietin (EPO) but are not induced in apoptosis following EPO withdrawal in EPO-dependent HCD57 cells

Jun N-terminal kinase (JNK) and p38, members of the mitogen-activated protein kinase family of serine/threonine kinases, are activated as a result of cellular stress but may also play a role in growth factor-induced proliferation and/or survival or differentiation of many cells. A recent report has implicated JNK and p38 in the induction of apoptosis in the erythropoietin (EPO)-dependent erythroid cell line HCD57 following EPO withdrawal, whereas our previously reported data did not support a role for JNK in growth factor withdrawal-induced apoptosis in HCD57 cells. Therefore, further testing was done to see if JNK was activated in EPO withdrawal-induced apoptosis; the study was extended to p38 and characterized the effect of EPO on JNK and p38 activities. Treatment of HCD57 cells with EPO resulted in a gradual and sustained activation of both JNK and p38 activity; these activities decreased on EPO withdrawal. Transient activation of p42/p44 extracellular signal-related kinases (ERK) was also detected. Inhibition of ERK activity inhibited proliferation in EPO-treated cells but neither induced apoptosis nor activated JNK. Inhibition of p38 activity inhibited proliferation but did not protect HCD57 cells from apoptosis induced by EPO withdrawal. Treatment of HCD57 cells with tumor necrosis factor-alpha induced JNK activation but did not induce apoptosis. These results implicate JNK, p38, and ERK in EPO-induced proliferation and/or survival of erythroid cells but do not support a role for JNK or p38 in apoptosis induced by EPO withdrawal from erythroid cells.     

Tyrosine kinase and phosphatidylinositol 3-kinase activation are required for cyclic adenosine 3',5'-monophosphate-dependent potentiation of deoxyribonucleic acid synthesis induced by insulin-like growth factor-I in FRTL-5 cells

In previous studies, we showed that pretreatment of rat FRTL-5 thyroid cells with TSH, or other agents that increased intracellular cAMP, markedly potentiated DNA synthesis in response to insulin-like growth factor-I (IGF-I). In addition, we found that TSH pretreatment caused an increase in tyrosine phosphorylation of intracellular proteins including an unidentified 125-kDa protein that was well correlated with the TSH-potentiating effect on DNA synthesis induced by IGF-I. These results suggested that cAMP amplified IGF-I-dependent signals for cell growth through changes of cAMP-dependent tyrosine phosphorylation. The present studies were undertaken to determine how tyrosine kinase activation followed by an increase in tyrosine phosphorylation is required for cAMP-dependent potentiation of DNA synthesis induced by IGF-I in this cell line. First of all, we measured tyrosine kinase or protein-tyrosine phosphatase activities in the cell lysates by the in vitro assay. Chronic treatment with TSH or (Bu)2-cAMP stimulated tyrosine kinase activity in the particulate fraction and protein-tyrosine phosphatase activity in the soluble fraction, suggesting that tyrosine kinase plays more important roles for a cAMP-dependent increase in tyrosine phosphorylation of intracellular proteins. The increased tyrosine kinase activity was sensitive to genistein, a potent tyrosine kinase inhibitor. Genistein abolished both the cAMP-dependent increase in tyrosine phosphorylation of the 125-kDa protein and the enhanced DNA synthesis induced by IGF-I in a similar concentration-dependent manner. The only tyrosine-phosphorylated protein associated with the p85 regulatory subunit of phosphatidylinositol (PI) 3-kinase in response to cAMP was 125 kDa. In addition, we found that PI 3-kinase activity bound to p85 subunit significantly increased after (Bu)2cAMP treatment. These results suggested that cAMP stimulates PI 3-kinase through tyrosine phosphorylation of the 125-kDa protein. We then measured DNA synthesis in cells pretreated for 24 h with TSH or (Bu)2cAMP in the absence or presence of LY294002, a PI 3-kinase inhibitor, followed by treatment with IGF-I for 24 h. Presence of LY294002 during TSH or (Bu)2cAMP pretreatment completely abolished cAMP-dependent potentiation of DNA synthesis induced by IGF-I. These results suggest that in FRTL-5 cells cAMP activates genistein-sensitive tyrosine kinases that in turn activate PI 3-kinase activity. These mechanisms appear to be necessary for cAMP-dependent potentiation of the DNA synthesis induced by IGF-I.     

Cyclic adenosine 3',5'-monophosphate (cAMP)-dependent protein kinases, but not exchange proteins directly activated by cAMP (Epac), mediate thyrotropin/cAMP-dependent regulation of thyroid cells

TSH, mainly acting through cAMP, is the principal physiological regulator of thyroid gland function, differentiation expression, and cell proliferation. Both cAMP-dependent protein kinases [protein kinase A (PKA)] and the guanine-nucleotide-exchange factors for Rap proteins, exchange proteins directly activated by cAMP (Epac) 1 and Epac2, are known to mediate a broad range of effects of cAMP in various cell systems. In the present study, we found a high expression of Epac1 in dog thyrocytes, which was further increased in response to TSH stimulation. Epac1 was localized in the perinuclear region. Epac2 showed little or no expression. The TSH-induced activation of Rap1 was presumably mediated by Epac1 because it was mimicked by the Epac-selective cAMP analog (8-p-chloro-phenyl-thio-2'-O-methyl-cAMP) and not by PKA-selective cAMP analogs. Surprisingly, in view of the high Epac1 expression and its TSH responsiveness, all the cAMP-dependent functions of TSH in cultures or tissue incubations of dog thyroid, including acute stimulation of thyroid hormone secretion, H(2)O(2) generation, actin cytoskeleton reorganization, p70(S6K1) activity, delayed stimulation of differentiation expression, and mitogenesis, were induced only by PKA-selective cAMP analogs. The Epac activator 8-p-chloro-phenyl-thio-2'-O-methyl-cAMP, used alone or combined with PKA-selective cAMP analogs, had no measurable effect on any of these TSH targets. Therefore, PKA activation seems to mediate all the recognized cAMP-dependent effects of TSH and is thus presumably responsible for the pathological consequences of its deregulation. The role of Epac1 and TSH-stimulated Rap1 activation in thyrocytes is still elusive.