Cyclic AMP regulates basement membrane heparan sulfate proteoglycan, perlecan, metabolism in rat glomerular epithelial cells.
Perlecan, the basement membrane heparan sulfate proteoglycan (HSPG), has been fully cloned from mouse and human tissues. When a cRNA probe of murine perlecan cDNA was employed in RNase protection assay to test whether rat glomerular epithelial cells (GEC) constitutively express perlecan, several bands of hybridization were seen, suggesting that sequences between rat and murine perlecan may not be identical. Using primers based on published cDNA sequences of murine and human perlecan and poly A+ RNA of rat GEC, we synthesized a 497 bp product (RPD-I) by RT-PCR. The deduced aminoacid sequence showed an 85% and 88% homology with domain I of murine and human perlecan, respectively. The three putative sites containing the consensus sequence SGD for attachment of heparan sulfate chains were fully conserved in the rat perlecan as was a site (NFT) for attachment of N-linked oligosaccharide. RPD-I detected a > 9.5 kb transcript of perlecan in RNA of GEC, similar in size to that present in rat glomeruli. Employing a riboprobe synthesized from RPD-I in RNase protection assay we examined whether dbcAMP regulated perlecan expression in the GEC. At 1, 6, 24 and 48 h of incubation, 1 mM dbcAMP caused 43%, 32%, 47% and 40% reduction in mRNA abundance of perlecan, respectively. Immunoprecipitation showed a corresponding reduction of 61%, 70% and 65% in the synthesis of 35SO4 labeled basement membrane HSPG by the GEC following 12, 24 and 48 h of incubation with dbcAMP. Following incubation for 1 and 24 h prostaglandins, PGE1 and PGE2 (1 uM), known activators of glomerular adenylate cyclase, reduced perlecan mRNA abundance to a similar extent as dbcAMP on northern analysis. Our results show that glomerular basement membrane HSPG synthesized by the GEC belongs to the perlecan family. Decrease of GEC perlecan gene expression and synthesis by cAMP and prostaglandins may be of relevance to proteinuric states characterized by activation of these mediators.
Ko
C W
CW
Department of Medicine, University of Texas Health Science Center, San Antonio 78284, USA.
Bhandari
B
B
Yee
J
J
Terhune
W C
WC
Maldonado
R
R
Kasinath
B S
BS
D000527:Alprostadil / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001485:Basement Membrane / Q000187:drug effects / Q000378:metabolism; D003994:Bucladesine / Q000494:pharmacology; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism*; D015232:Dinoprostone / Q000494:pharmacology; D004848:Epithelium / Q000187:drug effects / Q000378:metabolism; D005786:Gene Expression Regulation; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D007678:Kidney Glomerulus / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011509:Proteoglycans / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017386:Sequence Homology, Amino Acid
D000527:Alprostadil; D003994:Bucladesine; D000242:Cyclic AMP; D015232:Dinoprostone; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate; D011509:Proteoglycans; D012333:RNA, Messenger; C071927:perlecan