Heterogeneous Nuclear Ribonucleoprotein K Modulates Angiotensinogen Gene Expression in Kidney Cells

Chih-Chang Wei,Shao-Ling Zhang,Yun-Wen Chen,Deng-Fu Guo,Julie R Ingelfinger,Karol Bomsztyk,John S.D Chan

doi:10.1074/jbc.m601945200

Abstract

The present studies aimed to identify the 70-kDa nuclear protein that binds to an insulin-responsive element in the rat angiotensinogen gene promoter and to define its action on angiotensinogen gene expression. Nuclear proteins were isolated from rat kidney proximal tubular cells and subjected to two-dimensional electrophoresis. The 70-kDa nuclear protein was detected by Southwestern blotting and subsequently identified by mass spectrometry, which revealed that it was identical to 65-kDa heterogeneous nuclear ribonucleoprotein K (hnRNP K). hnRNP K bound to the insulin-responsive element of the rat angiotensinogen gene was revealed by a gel mobility shift assay and chromatin immunoprecipitation assay. hnRNP K inhibited angiotensinogen mRNA expression and promoter activity. In contrast, hnRNP K down-expression by small interference RNA enhanced angiotensinogen mRNA expression. Moreover, hnRNP K interacted with hnRNP F in pulldown and co-immunoprecipitation assays. Co-transfection of hnRNP K and hnRNP F further suppressed angiotensinogen mRNA expression. Finally, in vitro and in vivo studies demonstrated that high glucose increases and insulin inhibits hnRNP K expression in rat kidney proximal tubular cells. In conclusion, our experiments revealed that hnRNP K is a nuclear protein that binds to the insulin-responsive element of the rat angiotensinogen gene promoter and modulates angiotensinogen gene transcription in the kidney.

Highlights

Angiotensinogen (AGT), a glycoprotein consisting of 452 amino acid residues with an apparent molecular mass of 62–65 kDa, is the sole substrate in the renin-angiotensin system (RAS) cascade [13, 14]
We recently reported that the 48-kDa nuclear pro- in vitro and in vivo studies revealed that high glucose or hypertein is identical to the 46-kDa heterogeneous nuclear ribonucleo- glycemia increased and insulin inhibited heterogeneous nuclear ribonucleoprotein K (hnRNP K) expression in rat protein F [35]
Identification of 70-kDa insulin-responsive element (IRE)-binding Proteins (IRE-BPs) in IRPTCs—Fig. 1A shows the staining of nuclear proteins after two-dimensional electrophoresis

Summary

EXPERIMENTAL PROCEDURES

D(ϩ)-glucose, D-mannitol, and insulin were purchased from Sigma-Aldrich Canada Ltd. (Oakville, Canada). The bacterial expression vector pGex 4T-3 and mammalian expression vectors pcDNA 3.1 and pRC/RSV were purchased from Amersham Biosciences and Invitrogen, respectively. The plasmid containing the concatemeric wide type and mutant rAGT-IRE motif DNAs were constructed by inserting the double-stranded concatemeric wide type or mutant rAGTIRE motif oligonucleotide with the NotI enzyme restriction site added on both termini into the polyclonal site of pcDNA 3.1 by conventional methodology. The characteristics of IRPTCs, which express the mRNA and protein of rAGT, renin, angiotensin-converting enzyme, and angiotensin II receptors, have been described previously [41]. The other was electrotransferred to a Hybond C-extra hnRNP K fusion proteins were tested in GMSAs. membrane (Amersham Biosciences) for Southwestern blotting. Cross-linking was reversed by adding 1 ␮l of 10 mg/ml RNase and

M NaCl to a final concentration of

RESULTS

DISCUSSION