title: Regulation of mesangial cell alpha-smooth muscle actin expression in 3-dimensional matrix by high glucose and growth factors.
abstract: 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.
authors.affiliation: University Health Network, Toronto, Ont., Canada. catharine.whiteside@utoronto.ca
authors.forename: Catharine
authors.initials: C
authors.lastname: Whiteside
authors.forename: Snezana
authors.initials: S
authors.lastname: Munk
authors.forename: Eric
authors.initials: E
authors.lastname: Ispanovic
authors.forename: Hong
authors.initials: H
authors.lastname: Wang
authors.forename: Howard
authors.initials: H
authors.lastname: Goldberg
authors.forename: Andras
authors.initials: A
authors.lastname: Kapus
authors.forename: Ling
authors.initials: L
authors.lastname: Xia
mesh_terms: 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
chemical_list: D000199:Actins; D005947:Glucose; D036341:Intercellular Signaling Peptides and Proteins; D011493:Protein Kinase C; C498862:Acta2 protein, rat