Interaction between 4-Hydroxy-2,3-alkenals and the Platelet-derived Growth Factor-β Receptor: REDUCED TYROSINE PHOSPHORYLATION AND DOWNSTREAM SIGNALING IN HEPATIC STELLATE CELLS

Gaia Robino,Maurizio Parola,Fabio Marra,Alessandra Caligiuri,Raffaella M.S De Franco,Elena Zamara,Giorgio Bellomo,Paolo Gentilini,Massimo Pinzani,Mario U Dianzani

doi:10.1074/jbc.m007694200

Abstract

Hepatic stellate cells (HSC) undergo activation toward myofibroblast-like cells during early stages of liver injury associated with fibrogenesis. Platelet-derived growth factor (PDGF), particularly its BB isoform, has been identified as the most potent mitogen for HSC. 4-Hydroxy-2,3-nonenal and related 4-hydroxy-2, 3-alkenals (HAKs) have been suggested to modulate the process of HSC activation. In this study we investigated the relationship between HAKs and PDGF receptor activation in human HSC. By employing noncytotoxic concentrations (10(-6) m) of HAKs, we observed a significant inhibition of PDGF-BB-dependent DNA synthesis. HAKs inhibited relevant pathways of PDGF-BB-dependent mitogenic signaling, including autophosphorylation of PDGF receptor (PDGF-R) beta subunits and activation of phosphatidylinositol 3-kinase and extracellular regulated kinases 1/2. Inhibition of DNA synthesis was reversible, and recovery of PDGF-mediated mitogenic signaling occurred within 24-48 h and was associated with HAKs-induced up-regulation of PDGF-R beta gene expression. 4-Hydroxy-2,3-nonenal, used as a model HAK, inhibited the intrinsic tyrosine kinase activity associated with the PDGF-R beta subunit, whereas binding of PDGF to its receptor was unaffected. This study identifies a novel regulatory mechanism of reactive aldehydes on PDGF receptor signaling and biologic actions, which may be relevant in several pathophysiological conditions, including liver fibrosis.

Highlights

Hepatic stellate cells (HSC) undergo activation toward myofibroblast-like cells during early stages of liver injury associated with fibrogenesis
In chronic fibrogenic diseases, where Platelet-derived growth factor (PDGF) has been described to play a relevant role as a mitogenic factor, oxidative stress-related molecules are generated and have been suggested to be able to modulate the proliferative response of key myofibroblast-like target cells such as, for example, activated HSC in the case of liver fibrosis [43, 44]
We showed that the molecular basis for this effect is the inhibition of the intrinsic tyrosine kinase activity of PDGF receptor (PDGF-R) ␤ subunits and, as a consequence, the reduction of extracellular-signal regulated kinase (ERK), and phosphatidylinositol 3-kinase (PI 3-K) activity, two pathways known to play a key role in mediating the mitogenic effect of PDGF in HSC

Summary

EXPERIMENTAL PROCEDURES

Materials—4-Hydroxy-2,3-alkenals of different chain length, HNE, 4-hydroxy-2,3-octenal (HOE) and 4-hydroxy-2,3-undecenal (HUE), were kindly provided by Prof. The assay was performed resuspending the beads in kinase buffer (50 mM Hepes, pH 7.5, 100 mM NaCl, 10 mM MgCl2, 10 mM MnCl2, 10 mM ␤-glycerophosphate, and 0.5 mM sodium orthovanadate) in the presence of 1 ␮M protein kinase A inhibitor peptide, 50 ␮M unlabeled ATP, and 6 ␮Ci of [␥-32P]ATP, using exogenous histone H2B (1.5 ␮g/assay tube) as the substrate and incubating for 20 min at room temperature. The immunoprecipitated proteins were washed twice in lysis buffer and once in 50 mM Tris-HCl, pH 7.4, containing 1 mM sodium orthovanadate and resuspended in 50 mM Hepes at pH 7.4 At this point, 1 ␮M HNE was added to one aliquot of samples A and C for 15 min to establish the effects of HNE on purified PDGF-R ␤ subunits in vitro. Statistical analysis was performed by Student’s t test (p Յ 0.05 was considered significant)

RESULTS

87 Ϯ 5 23 Ϯ 6a

DISCUSSION