Expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-1 and -2 and its unique alternative splice variants in U87 glioma cells with ERN1 loss of function

Was received 15.07.2011 The endoplasmic reticulum–nuclei-1 (ERN1) sensing and signaling enzyme mediates a set of complex intracellular signaling events known as the unfolded protein response. We have studied the effect of hypoxia and ischemic conditions (glucose or glutamine deprivation) on the expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-1 and -2 (PFKFB1 and PFKFB2) genes in glioma U87 cells and its subline with suppressed function of ERN1 sensing and signaling enzyme. We have identified three unique alternative splice variants of PFKFB2 and PFKFB1 with insert or deletions in its N-terminal region which eliminate 6-phosphofructo-2-kinase activity. It was shown that blockade of ERN1 enzyme function, the key endoplasmic reticulum stress sensor, leads to an increase in the expression levels of PFKFB1 and PFKFB2 mRNAs as well as its alternative splice variants. Moreover, the expression level of PFKFB2 mRNA increases both in control glioma cells and decreases in genetically modified glioma cells treated by hypoxia. At the same time, expression level of PFKFB1 mRNA and its alternative splice variants does not change significantly at this experimental condition. Exposure cells under glutamine or glucose deprivation conditions leads to increase the expression level of PFKFB1 and PFKFB2 mRNA in control glioma cells only, but expression level of alternative splice variants of these mRNA also increases in control glioma cells and only in glutamine deprivation condition. It was shown also that blockade of ERN1 signaling enzyme function eliminate the increase of PFKFB1 and PFKFB2 mRNA as well as its alternative splice variants induced in glioma cells by glutamine and glucose deprivation conditions. Thus, results of this study clearly demonstrate that the expression level of PFKFB1 and PFKFB2 as well as its alternative splice variants without 6-phosphofructo-2-kinase activity increases in glioma cells with ERN1 signaling enzyme loss of function and that glutamine and glucose deprivation conditions also lead to increase the expression level of these PFKFB but in control glioma cells only. It is possible that increase of the expression level of PFKFB1 and PFKFB2 genes in glutamine and glucose deprivation conditions is mediated by ERN1 signaling system because it miss in glioma cells with ERN1 loss of function and is observed in control glioma cells only. Ключові слова: mRNA expression, PFKFB1, PFKFB2, unique alternative splice variants, glioma cells, endoplasmic reticulum–nuclei-1 (ERN1), hypoxia, glucose and glutamine deprivation.