Chloride intracellular channel 1 regulates osteoblast differentiation.
We have identified chloride intracellular channel 1 (CLIC1) through proteomic approach, which was increased in response to canonical wnt signaling while being almost shut-off by adipogenic treatment in mouse mesenchymal C3H10T1/2 cells. We found that CLIC1 was expressed in mouse (MC3T3-E1), rat (ROS 17/2.8 and UMR-106) or human (MG63 and SaOS2) osteoblastic cell lines as well as primary culture of mouse calvarial cells by RT-PCR or Western blot analysis. The expression level of CLIC1 is increased upon treatment of osteogenic medium, whereas it almost disappeared in adipogenic condition, confirming the proteomic data. The expression of CLIC1 was localized mainly in nuclear membrane and vesiculo-cytoplasmic, the latter of which was colocalized with mitochondria. Retroviral overexpression of CLIC1 did not increase whole-cell current but induces hyperpolarization of mitochondrial membrane potential estimated using the fluorescent dye TMRE. Moreover, overexpression of CLIC1 resulted in increase in osteoblastic differentiation of C3H10T1/2 cells as measured by ALP activities or osteoblastic gene expression (osterix, ALP and osteocalcin), although it did not result in induction of Runx2 transcription activities at mouse osteocalcin (OG2) promoter. Finally, in vitro knock-down of CLIC1 using stable siRNA CLIC1 significantly suppressed osteoblastic differentiation. Taken together, these results suggest that CLIC1 may play a role in the regulation of osteoblastic differentiation from mesenchymal progenitors, although its physiologic role in osteoblasts remains to be determined.
Yang
Jae-Yeon
JY
Department of Internal Medicine, Seoul National University College of Medicine, 101 Daehak-Ro, Jongno-Gu, Seoul 110-744, Korea.
Jung
Ju Yeon
JY
Cho
Sun Wook
SW
Choi
Hyung Jin
HJ
Kim
Sang Wan
SW
Kim
Seong Yeon
SY
Kim
Hee Joong
HJ
Jang
Chang Han
CH
Lee
Min Goo
MG
Han
Jin
J
Shin
Chan Soo
CS
D050156:Adipogenesis; D000469:Alkaline Phosphatase / Q000037:antagonists & inhibitors / Q000378:metabolism; D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D018118:Chloride Channels / Q000235:genetics / Q000378:metabolism*; D003470:Culture Media; D015180:Electrophoresis, Gel, Two-Dimensional; D005786:Gene Expression Regulation; D006801:Humans; D053078:Membrane Potential, Mitochondrial; D051379:Mice; D010006:Osteoblasts / Q000166:cytology* / Q000378:metabolism*; D010012:Osteogenesis; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D012190:Retroviridae / Q000235:genetics; D015398:Signal Transduction; D013347:Subcellular Fractions / Q000378:metabolism; D051153:Wnt Proteins / Q000378:metabolism
C501966:CLIC1 protein, human; C546620:CLIC1 protein, rat; D018118:Chloride Channels; C501967:Clic1 protein, mouse; D003470:Culture Media; D034741:RNA, Small Interfering; D051153:Wnt Proteins; D000469:Alkaline Phosphatase