Intracellular calcium mobilization induces immediate early gene pip92 via Src and mitogen-activated protein kinase in immortalized hippocampal cells

Regulation of intracellular calcium levels plays a central role in cell survival, proliferation, and differentiation. A cell-permeable, tumor-promoting thapsigargin elevates the intracellular calcium levels by inhibiting endoplasmic reticulum Ca(2+)-ATPase. The Src-tyrosine kinase family is involved in a broad range of cellular responses ranging from cell growth and cytoskeletal rearrangement to differentiation. The immediate early gene pip92 is induced in neuronal cell death as well as cell growth and differentiation. To resolve the molecular mechanism of cell growth by intracellular calcium mobilization, we have examined the effect of thapsigargin and subsequent intracellular calcium influx on pip92 expression in immortalized rat hippocampal H19-7 cells. An increase of intracellular calcium ion levels induced by thapsigargin stimulated the expression of pip92 in H19-7 cells. Transient transfection of the cells with kinase-inactive mitogen-activated protein kinase kinase (MEK) and Src kinase or pretreatment with the chemical MEK inhibitor PD98059 significantly inhibited pip92 expression induced by thapsigargin. When constitutively active v-Src or MEK was overexpressed, the transcriptional activity of the pip92 gene was markedly increased. Dominant inhibitory Raf-1 blocked the transcriptional activity of pip92 induced by thapsigargin. The transcription factor Elk1 is activated during thapsigargin-induced pip92 expression. Taken together, these results suggest that an increase of intracellular calcium ion levels by thapsigargin stimulates the pip92 expression via Raf-MEK-extracellular signal-regulated protein kinase- as well as Src kinase-dependent signaling pathways.ope

Darier병 관련 sarco/endoplasmic reticulum Ca2+-ATPase 변이에 의한

Dept. of Medical Science/박사[한글] Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump는 세포질의 칼슘을 세포내 저장소로 이동시켜 세포질의 칼슘농도가 낮게 유지시키고 여러 자극에 의하여 세포 칼슘신호가 일어날 수 있게 하는데 중요한 역할을 하고 있다. 최근 제2형 Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2) pump를 발현하는 ATP2A2에 유전적 변이가 생기면 Darier병이 발생함이 밝혀졌다. Darier병은 상염색체 우성으로 유전되는 피부질환으로 이 질환의 피부병리 소견은 각질세포 사이의 세포간결합을 잃는 acantholysis와 비정상적인 각질화이다. 본 연구에서는 Darier병 환자에서 발견한 12개의 돌연변이 SERCA를 HEK 293 세포주에 transfection하여 여러 성질을 규명하였다. 각 돌연변이 유전자는 다양한 양의 단백질을 발현하였고, 이는 부분적으로는 proteasome에 의한 단백분해 때문에 일어난 현상이었다. 모든 돌연변이 SERCA는 야생형 SERCA보다 작은 활성을 보였으며 다섯개의 돌연변이 SERCA는 HEK 293 세포주의 내재한 SERCA혹은 같이 transfection한 야생형 SERCA를 억제하였다. 작은 SERCA 활성을 가진 돌연변이에서는 내형질세망의 칼슘도 적었다. 이러한 억제의 가능한 기전을 규명하고자 SERCA의 passive leak, IP3 수용체의 감수성 및 야생형 SERCA의 단백 발현을 측정하였으나 돌연변이 SERCA가 이러한 차이를 유발하지는 않았다. 그러나 면역침전반응으로 두개의 SERCA 단백질이 서로 결합한다는 것을 발견하였으며 이는 돌연변이 SERCA가 야생형 SERCA에 결합하여 그 기능을 억제하고 있을 가능성이 높음을 뜻한다. 본 연구의 결과 SERCA가 dimer 혹은 그 이상의 단백복합체로 작용하고 있으며 SERCA에 의해 고농도로 유지되는 내형질세망 칼슘이 세포간결합에 있어서 중요한 역할을 한다. [영문] Darier''s disease (DD) is an autosomal dominant skin disorder characterized by loss of adhesion between epidermal cells (acantholysis) and abnormal keratinization. Recently, mutations in the ATP2A2 gene encoding a Ca2+-ATPase pump of the sarco endoplasmic reticulum (SERCA2) have been discovered as the cause of DD. In the present work twelve DD-causing SERCA2b mutations were selected to study the underlying pathologic mechanisms of DD and to elucidate the role of SERCA2b in calcium signaling. Most mutations markedly affected protein expression, partially due to enhanced proteasome-mediated degradation. When SERCA activity was measured in mutant-transfected cells, all DD-causing mutants showed lower activity than the wild type SERCA2b. Several mutants that cause relatively severe phenotype of DD inhibited endogenous and the co-transfected wild type SERCA2b activity. These effects were not due to changes in passive leak, IP3 receptor activity or sensitivity to IP3. In co-immunoprecipitation experiments, SERCA2b monomers interact to influence the activity of each other. These findings reveal the importance of SERCA2b dimerization and ER Ca2+ in physiologic cellular function and pathogenesis of DD.ope

Ca2+ Activates Cystic Fibrosis Transmembrane Conductance Regulator- and Cl−-dependent HCOFormula Transport in Pancreatic Duct Cells

Pancreatic duct cells secrete bicarbonate-rich fluids, which are important for maintaining the patency of pancreatic ductal trees as well as intestinal digestive function. The bulk of bicarbonate secretion in the luminal membrane of duct cells is mediated by a Cl(-)-dependent mechanism (Cl(-)/HCO(3)(-) exchange), and we previously reported that the mechanism is CFTR-dependent and cAMP-activated (Lee, M. G., Choi, J. Y., Luo, X., Strickland, E., Thomas, P. J., and Muallem, S. (1999) J. Biol. Chem. 274, 14670-14677). In the present study, we provide comprehensive evidence that calcium signaling also activates the same CFTR- and Cl(-)-dependent HCO(3)(-) transport. ATP and trypsin evoked intracellular calcium signaling in pancreatic duct-derived cells through the activation of purinergic and protease-activated receptors, respectively. Cl(-)/HCO(3)(-) exchange activity was measured by recording pH(i) in response to [Cl(-)](o) changes of the perfusate. In perfusate containing high concentrations of K(+), which blocks Cl(-) movement through electrogenic or K(+)-coupled pathways, ATP and trypsin highly stimulated luminal Cl(-)/HCO(3)(-) exchange activity in CAPAN-1 cells expressing wild-type CFTR, but not in CFPAC-1 cells that have defective (DeltaF508) CFTR. Notably, adenoviral transfection of wild-type CFTR in CFPAC-1 cells completely restored the stimulatory effect of ATP on luminal Cl(-)/HCO(3)(-) exchange. In addition, the chelation of intracellular calcium by 1,2-bis(2-aminophenoxy)ethane-N,N,N,N'-tetraacetic acid (BAPTA) treatment abolished the effect of calcium agonists on luminal Cl(-)/HCO(3)(-) exchange. These results provide a molecular basis for calcium-induced bicarbonate secretion in pancreatic duct cells and highlight the importance of CFTR in epithelial bicarbonate secretion induced by various stimuli.ope