ABSTRACT
AIMS/HYPOTHESIS: Human adipose tissue macrophages (ATMs) display an alternatively activated (M2) phenotype, but are still able to produce excessive inflammatory mediators. However, the processes driving this particular ATM phenotype are not understood. Genome-wide association studies associated the CDKN2A locus, encoding the tumour suppressor p16(INK4A), with the development of type 2 diabetes. In the present study, p16(INK4A) levels in human ATMs and the role of p16(INK4A) in acquiring the ATM phenotype were assessed. METHODS: Gene expression of p16 ( INK4A ) in ATMs was analysed and compared with that in monocyte-derived macrophages (MDMs) from obese patients or with macrophages from human atherosclerotic plaques (AMs). Additionally, p16(INK4A) levels were studied during macrophage differentiation and polarisation of monocytes isolated from healthy donors. The role of p16(INK4A) in MDMs from healthy donors was investigated by small interfering (si)RNA-mediated silencing or adenovirus-mediated overproduction of p16(INK4A). RESULTS: Compared with MDMs and AMs, ATMs from obese patients expressed lower levels of p16 ( INK4A ). In vitro, IL-4-induced M2 polarisation resulted in lower p16(INK4A) protein levels after differentiation of monocytes from healthy donors in macrophages. Silencing of p16(INK4A) in MDMs mediated by siRNA increased the expression of M2 marker genes and enhanced the response to lipopolysaccharide (LPS), to give a phenotype resembling that of ATM. By contrast, adenovirus-mediated overproduction of p16(INK4A) in MDMs diminished M2 marker gene expression and the response to LPS. Western blot analysis revealed that p16(INK4A) overproduction inhibits LPS- and palmitate-induced Toll-like receptor 4 (TLR4)-nuclear factor of κ light polypeptide gene enhancer in B cells (NF-κB) signalling. CONCLUSIONS/INTERPRETATION: These results show that p16(INK4A) inhibits the acquisition of the ATM phenotype. The age-related increase in p16(INK4A) level may thus influence normal ATM function and contribute to type 2 diabetes risk.
Subject(s)
Adipose Tissue/metabolism , Cell Polarity , Cyclin-Dependent Kinase Inhibitor p16/biosynthesis , Macrophages/metabolism , Cyclin-Dependent Kinase Inhibitor p16/genetics , Diabetes Mellitus, Type 2/metabolism , Down-Regulation , Female , Gene Silencing , Humans , Male , NF-kappa B/metabolism , Obesity/metabolism , Plaque, Atherosclerotic/metabolism , RNA, Small Interfering/metabolism , Toll-Like Receptor 4/metabolismABSTRACT
The scattering of Madin-Darby canine kidney (MDCK) epithelial cells by scatter factor/hepatocyte growth factor (SF/HGF) is associated with transcriptional induction of the urokinase gene, which occurs essentially through activation of an EBS/AP1 response element. We have investigated the signal transduction pathways leading to this transcriptional response. We found that SF/HGF induces rapid and sustained phosphorylation of the extracellular signal-regulated kinase (ERK) MAPK while stimulating weakly and then repressing phosphorylation of the JUN N-terminal kinase (JNK) MAPK for several hours. This delayed repression of JNK was preceded by phosphorylation of the MKP2 phosphatase, and both MKP2 induction and JNK dephosphorylation were under the control of MEK, the upstream kinase of ERK. ERK and MKP2 stimulate the EBS/AP1-dependent transcriptional response to SF/HGF, but not JNK, which inhibits this response. We further demonstrated that depending on cell density, the RAS-ERK-MKP2 pathway controls this transrepressing effect of JNK. Together, these data demonstrate that in a sequential manner SF/HGF activates ERK and MKP2, which in turn dephosphorylates JNK. This sequence of events provides a model for efficient cell scattering by SF/HGF at low cell density.
Subject(s)
Hepatocyte Growth Factor/metabolism , Mitogen-Activated Protein Kinases/metabolism , Animals , Cell Division/drug effects , Cell Division/physiology , Cells, Cultured , Dogs , Enzyme Activation , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Hepatocyte Growth Factor/pharmacology , JNK Mitogen-Activated Protein Kinases , Kidney/cytology , Phosphorylation , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Tyrosine Phosphatases/metabolism , Transcription Factor AP-1/genetics , Transcription Factor AP-1/metabolism , Transcription, Genetic , ras Proteins/genetics , ras Proteins/metabolismABSTRACT
The scatter factor/hepatocyte growth factor regulates scattering and morphogenesis of epithelial cells through activation of the MET tyrosine kinase receptor. In particular, the noncatalytic C-terminal tail of MET contains two autophosphorylation tyrosine residues, which form a multisubstrate-binding site for several cytoplasmic effectors and are thought to be essential for signal transduction. We show here that a MET receptor mutated on the four C-terminal tyrosine residues, Y1311F, Y1347F, Y1354F, and Y1363F, can induce efficiently a transcriptional response and cell scattering, whereas it cannot induce cell morphogenesis. Although the mutated receptor had lost its ability to recruit and/or activate known signaling molecules, such as GRB2, SHC, GAB1, and PI3K, by using a sensitive association-kinase assay we found that the mutated receptor can still associate and phosphorylate a approximately 250-kDa protein. By further examining signal transduction mediated by the mutated MET receptor, we established that it can transmit efficient RAS signaling and that cell scattering by the mutated MET receptor could be inhibited by a pharmacological inhibitor of the MEK-ERK (MAP kinase kinase-extracellular signal-regulated kinase) pathway. We propose that signal transduction by autophosphorylation of the C-terminal tyrosine residues is not the sole mechanism by which the activated MET receptor can transmit RAS signaling and cell scattering.
