Profiling of human mesangial cell subproteomes reveals a role for calmodulin in glucose uptake.
Proteomics combined with cell fractionation was used to identify proteins regulated by high glucose (HG) in human mesangial cells (HMC). Total membrane and cytosolic fraction proteins derived from HMC after 7 days of HG exposure were resolved by a two-dimensional gel electrophoresis approach. DeCyder software was used to analyze the HG-induced protein spot dysregulation. In the membrane subproteome, of the 92 spots that were matched across all gels, HG induced significant downregulation of only 4 protein spots. The dysregulated spots from the membrane subproteome included binding protein (BiP), calreticulin precursor protein, a 63-kDa transmembrane protein from a ER/Golgi intermediate, and beta-subunit of collagen proline 4-hydroxylase. In the cytosolic subproteome, of the 122 spots that were matched across all gels, HG induced downregulation of 3 protein spots and upregulation of 2 protein spots significantly. Enolase 1, annexin VI, and gamma(2)-actin were decreased, whereas heat shock protein-70 kDa and calmodulin (CaM) were increased. Further confocal microscopy and Western immunoblotting of mesangial cells validated the increase in CaM. Immunoblotting of diabetic mouse and rat kidneys exhibited a marked increase in CaM at both early and late stages of diabetes, reflecting the potential physiological relevance of CaM upregulation. CaM-specific inhibitors blocked glucose transport stimulated by transforming growth factor-beta and insulin in mesangial cells. In conclusion, using a combination of cell fractionation and protein expression profiling, we identified a cohort of HG-dysregulated proteins in the HMC and identified a critical and as yet unrecognized role for CaM in glucose transport in mesangial cells.
Ramachandra Rao
Satish P
SP
Dept. of Medicine, Center for Novel Therapies for Kidney Disease, Suite 365 Jefferson Alumni Hall, 1020 Locust St., Philadelphia, PA 19107, USA. satish.rao@jefferson.edu
Wassell
Richard
R
Shaw
M Alexander
MA
Sharma
Kumar
K
D000818:Animals; D002147:Calmodulin / Q000502:physiology*; D003921:Diabetes Mellitus, Experimental / Q000503:physiopathology; D003922:Diabetes Mellitus, Type 1 / Q000503:physiopathology; D015536:Down-Regulation; D020869:Gene Expression Profiling; D005947:Glucose / Q000008:administration & dosage / Q000378:metabolism*; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D050527:Mesangial Cells / Q000378:metabolism*; D051379:Mice; D020543:Proteome / Q000032:analysis*; D051381:Rats; D016212:Transforming Growth Factor beta / Q000494:pharmacology
D002147:Calmodulin; D007328:Insulin; D020543:Proteome; D016212:Transforming Growth Factor beta; D005947:Glucose