Regulation of mesangial cell alpha-smooth muscle actin expression in 3-dimensional matrix by high glucose and growth factors.
OBJECTIVE
We postulated that alpha-smooth muscle actin expressed in primary cultured mesangial cells is down-regulated in 3-dimensional (D) culture and up-regulated by high glucose and growth factors.


METHODS
Primary rat mesangial cells were growth-arrested in 5.6 mM (NG) or 30 mM (HG) glucose for 14 days in 3-D Matrigel. Alpha-SM actin expression was analyzed by immunoblotting, real-time PCR and by alpha-SM actin promoter activity in response to 24 h stimulation with endothelin-1 (ET-1), angiotensin II (Ang II) or HG.


RESULTS
Alpha-SM actin mRNA, protein and promoter activity were reduced to significantly lower levels in 3-D cells compared to cells in 2-D. Up-regulation of alpha-SM expression was stimulated by ET-1, Ang II and HG. Specific inhibitors of protein kinase C (PKC)-alpha, -beta or -zeta prevented alpha-SM upregulation in HG. In NG, PKC and ERK1/2 activation were required for alpha-SM actin accumulation in 3-D in response to ET-1 or Ang II. In HG, enhanced expression of alpha-SM actin in response to ET-1 or Ang II was unchanged during PKC or ERK1/2 inhibition.


CONCLUSIONS
Mesangial cells in 3-D express low levels of alpha-SM actin representing a more differentiated state. Regulation of alpha-SM actin expression is dependent on specific PKC isozyme and ERK1/2 signaling.
Whiteside
Catharine
C
University Health Network, Toronto, Ont., Canada. catharine.whiteside@utoronto.ca
Munk
Snezana
S
Ispanovic
Eric
E
Wang
Hong
H
Goldberg
Howard
H
Kapus
Andras
A
Xia
Ling
L
D000199:Actins / Q000378:metabolism*; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002478:Cells, Cultured; D005109:Extracellular Matrix / Q000737:chemistry / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000502:physiology; D005947:Glucose / Q000378:metabolism*; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism*; D020935:MAP Kinase Signaling System / Q000502:physiology*; D050527:Mesangial Cells / Q000166:cytology / Q000502:physiology*; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley
D000199:Actins; D005947:Glucose; D036341:Intercellular Signaling Peptides and Proteins; D011493:Protein Kinase C; C498862:Acta2 protein, rat