Hepatocyte Growth Factor Suppresses Proinflammatory NFκB Activation through GSK3β Inactivation in Renal Tubular Epithelial Cells

Yan Ge,Lance D Dworkin,Abdalla Rifai,Rujun Gong,Shan Chen

doi:10.1074/jbc.m710396200

Yan Ge, Lance D Dworkin + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m710396200

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Abstract

Activation of NFkappaB is a fundamental cellular event central to all inflammatory diseases. Hepatocyte growth factor (HGF) ameliorates both acute and chronic inflammation in a multitude of organ systems through modulating NFkappaB activity; nevertheless, the exact molecular mechanism remains uncertain. Here we report that HGF through inactivation of GSK3beta suppresses NFkappaB p65 phosphorylation specifically at position Ser-468. The Ser-468 of RelA/p65 situates in a GSK3beta consensus motif and could be directly phosphorylated by GSK3beta both in vivo and in vitro, signifying Ser-468 of RelA/p65 as a putative substrate for GSK3beta. In addition, the C terminus of RelA/p65 harbors a highly conserved domain homologue of the consensus docking sequence for GSK3beta. Moreover, this domain was required for efficient phosphorylation of Ser-468 and was indispensable for the physical interaction between RelA/p65 and GSK3beta. HGF substantially intercepted this interaction by inactivating GSK3beta. Functionally, phosphorylation of Ser-468 of RelA/p65 was required for the induced expression of a particular subset of proinflammatory NFkappaB-dependent genes. Diminished phosphorylation at Ser-468 by HGF resulted in a gene-specific inhibition of these genes' expression. The action of HGF on proinflammatory NFkappaB activation was consistently mimicked by a selective GSK3beta inhibitor or GSK3beta knockdown by RNA interference but largely abrogated in cells expressing the mutant uninhibitable GSK3beta. Collectively, our findings suggest that HGF has a potent suppressive effect on NFkappaB activation, which is mediated by GSK3beta, an important signaling transducer controlling RelA/p65 phosphorylation specificity and directing the transcription of selective proinflammatory cytokines implicated in inflammatory kidney disease.

Highlights

Activation and regulation of NF␬B is a complex and highly orchestrated process, controlled by many signal transducers [23, 24], including GSK3␤ [25, 26]
GSK3␤ regulates NF␬B target gene expression in a gene-specific manner [32]. Consistent with this hypothesis, we demonstrated that GSK3␤ inactivation is required for Hepatocyte growth factor (HGF) suppression of multiple NF␬B-dependent proinflammatory chemokines and adhesion molecules [12, 30], strongly suggesting that GSK3␤ is involved in the anti-inflammatory action of hemagglutinin; WT, wild type; PBS, phosphate-buffered saline; small interference RNA (siRNA), small interfering RNA; RT, reverse transcription; EV, empty vector; TAD, transactivation domain
Suppression of NF␬B Activity by HGF Is Associated with Inhibitory Phosphorylation of GSK3␤—Our previous studies [20, 30] demonstrated that HGF ameliorates renal inflammation in animal models with chronic kidney disease and suppresses renal expression of multiple proinflammatory molecules, including MCP-1 and RANTES, two potent chemoattractant cytokines involved in the recruitment of mononuclear leukocytes to an inflammatory infiltrate

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—Human proximal tubular epithelial cells (HKC-8) (courtesy of Dr Racusen of John Hopkins University, Baltimore, MD) were maintained in Dulbecco’s modified Eagle’s medium/F-12 supplemented with 5% fetal bovine serum. Samples with equal amounts of total protein (50 ␮g/ml) were fractionated by 7.5–15% SDS-polyacrylamide gels under reducing conditions and analyzed by Western immunoblot as described previously [12]. After transfection with equal amounts of expression plasmid or empty vector pcDNA3 (Invitrogen), HKC cells were subjected to different treatments as indicated. HKC cells were cultured to 70% confluence and subjected to transfection of the reporter construct pGL-3␬B-Luc by using the Lipofectamine 2000. In Vitro Kinase Assay—Recombinant GSK3␤ (500 units; Cell Signaling Technology) was incubated with 50 ng of GST-p65 fusions proteins in 1ϫ GSK3␤ reaction buffer supplemented with 135 ␮M ATP in a total volume of 30 ␮l for 30 min at 30 °C.

RANTES Forward Reverse

RESULTS

DISCUSSION