Cw. Ko et al., CYCLIC-AMP REGULATES BASEMENT-MEMBRANE HEPARAN-SULFATE PROTEOGLYCAN, PERLECAN, METABOLISM IN RAT GLOMERULAR EPITHELIAL-CELLS, Molecular and cellular biochemistry, 162(1), 1996, pp. 65-73
Perlecan, the basement membrane heparan sulfate proteoglycan (HSPG), h
as been fully cloned from mouse and human tissues. When a cRNA probe o
f murine perlecan cDNA was employed in RNase protection assay to test
whether rat glomerular epithelial cells (GEC) constitutively express p
erlecan, several bands of hybridization were seen, suggesting that seq
uences between rat and murine perlecan may not be identical. Using pri
mers based on published cDNA sequences of murine and human perlecan an
d polyA+ 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 wi
th domain I of murine and human perlecan, respectively. The three puta
tive sites containing the consensus sequence SGD for attachment of hep
aran 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 th
at present in rat glomeruli. Employing a riboprobe synthesized from RP
D-I in RNase protection assay we examined whether dbcAMP regulated per
lecan 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 per
lecan, respectively. Immunoprecipitation showed a corresponding reduct
ion of 61%, 70% and 65% in the synthesis of (SO4)-S-35 labeled basemen
t membrane HSPG by the GEC following 12, 24 and 48 h of incubation wit
h dbcAMP. Following incubation for 1 and 24 h prostaglandins, PGE1 and
PGE2 (1 uM), known activators of glomerular adenylate cyclase, reduce
d perlecan mRNA abundance to a similar extent as dbcAMP on northern an
alysis. Our results show that glomerular basement membrane HSPG synthe
sized by the GEC belongs to the perlecan family. Decrease of GEC perle
can gene expression and synthesis by cAMP and prostaglandins may be of
relevance to proteinuric states characterized by activation of these
mediators.