Multiple phosphorylation sites of Drp1 have been characterized for their functional importance. However, the functional consequence of GSK3beta-mediated phosphorylation of Drp1 remains unclear. In this report, we pinpointed 11 Serine/Threonine sites spanning from residue 634∼736 of the GED domain and robustly confirmed Drp1 Ser693 as a novel GSK3beta phosphorylation site. Our results suggest that GSK3beta-mediated phosphorylation at Ser693 does cause a dramatic decrease of GTPase activity; in contrast, GSK3beta-mediated phosphorylation at Ser693 appears not to affect Drp1 inter-/intra-molecular interactions. After identifying Ser693 as a GSK3beta phosphorylation site, we also determined that K679 is crucial for GSK3beta-binding, which strongly suggests that Drp1 is a novel substrate for GSK3beta. Thereafter, we found that overexpressed S693D, but not S693A mutant, caused an elongated mitochondrial morphology which is similar to that of K38A, S637D and K679A mutants. Interestedly, using H89 and LiCl to inhibit PKA and GSK3beta signaling, respectively, it appears that a portion of the elongated mitochondria switched to a fragmented phenotype. In investigating the biofunctionality of phosphorylation sites within the GED domain, cells overexpressing Drp1 S693D and S637D, but not S693A, showed an acquired resistance to H2O2-induced mitochondrial fragmentation and ensuing apoptosis, which affected cytochrome c, capase-3, -7, and PARP, but not LC3B, Atg-5, Beclin-1 and Bcl2 expressions. These results also showed that the S693D group is more effective in protecting both non-neuronal and neuronal cells from apoptotic death than the S637D group. Altogether, our data suggest that GSK3beta-mediated phosphorylation at Ser693 of Drp1 may be associated with mitochondrial elongation via down-regulating apoptosis, but not autophagy upon H2O2 insult.

No abstract

Diabetic nephropathy (DN) is characterized by glomerulopathy and tubulointerstitial expansion followed by renal fibrosis. Angiotensin II (Ang II) and connective tissue growth factor (CTGF) are involved in the pathogenesis of DN, while Janus kinase 2 (JAK2) is important in advanced glycation end-product (AGE)-induced effects in renal interstitial (NRK-49F) fibroblasts. Thus, we studied the role of Ang II, CTGF, and JAK2 in AGE-induced effects in NRK-49F cells. We found that AGE (150 microg/ml) increased mitogenesis and type I collagen production at 7 days while Ang II (10(-7)M) increased mitogenesis and type I collagen production at 3 days. We also found that AGE (150 microg/ml) increased angiotensinogen protein at 2 days, which was attenuated by AG-490 (a JAK2 inhibitor). AGE (150 microg/ml) increased CTGF mRNA and protein expression at 3 and 5 days, respectively. Ang II (10(-7)M) increased CTGF mRNA and protein expression at 1 and 2 days, respectively, which were attenuated by AG-490. Moreover, losartan (a type I angiotensin receptor blocker) and captopril (an angiotensin converting enzyme inhibitor) attenuated AGE-induced CTGF mRNA/protein expression while attenuating AGE-induced mitogenesis and type I collagen production. AG-490 and CTGF antisense (but not sense) oligodeoxynucleotide (ODN) attenuated Ang II (10(-7)M) and AGE-induced mitogenesis and type I collagen production at 3 and 7 days, respectively. We concluded that AGE (150 microg/ml)-induced mitogenesis and type I collagen production are dependent on the Ang II-JAK2-CTGF pathway in NRK-49F cells. Moreover, Ang II-induced mitogenesis and type I collagen production are dependent on the JAK2-CTGF pathway.

No abstract

Previously, we showed that Janus kinase 2 (JAK2) is important in advanced glycation end-product (AGE)-induced effects in renal interstitial (NRK-49F) fibroblasts. Leptin is a JAK2-activating cytokine via the long form leptin receptor (Ob-Rb). Leptin and connective tissue growth factor (CTGF) may be involved in renal fibrosis. However, the relationship between leptin and CTGF in terms of AGE-induced effects remains unknown. Thus, the effects of AGE (150 microg/ml) and leptin on mitogenesis, CTGF and collagen expression in NRK-49F cells were determined. We found that leptin and AGE increased mitogenesis and type I collagen protein expression at 3 and 7 days, respectively. AGE increased leptin mRNA and protein expression at 2-3 days. AGE increased CTGF mRNA and protein expression at 3-5 days. AG-490 (JAK2 inhibitor) abrogated AGE-induced leptin mRNA and protein expression at 2-3 days. AG-490 and Ob-Rb anti-sense oligodeoxynucleotides (ODN) abrogated AGE-induced CTGF mRNA and protein expression at 3-5 days. AG-490 and CTGF anti-sense ODN abrogated AGE-induced mitogenesis and collagen protein expression at 7 days. Additionally, leptin dose (0.2-1 microg/ml) and time (1-2 days)-dependently increased CTGF protein expression. AG-490 abrogated leptin (1 microg/ml)-induced CTGF protein expression at 2 days. AG-490 and CTGF anti-sense ODN abrogated leptin-induced mitogenesis and collagen protein expression at 3 days. We concluded that AGE induced JAK2 to increase leptin while leptin induced JAK2 to increase CTGF-induced mitogenesis and type I collagen protein expression in NRK-49F cells. Additionally, AGE-induced mitogenesis and type I collagen protein expression were dependent on leptin-induced CTGF.