Activated α2 macroglobulin induces matrix metalloproteinase 9 expression by low‐density lipoprotein receptor‐related protein 1 through MAPK‐ERK1/2 and NF‐κB activation in macrophage‐derived cell lines

Macrophages under certain stimuli induce matrix metalloproteinase 9 (MMP‐9) expression and protein secretion through the activation of MAPK‐ERK and NF‐κB signaling pathways. Previously, we demonstrated that activated α₂‐macroglulin (α₂M*) through the interaction with its receptor low‐density lipoprotein receptor‐related protein 1 (LRP1) induces macrophage proliferation mediated by the activation of MAPK‐ERK1/2. In the present work, we examined whether α₂M*/LRP1interaction could induce the MMP‐9 production in J774 and Raw264.7 macrophage‐derived cell lines. It was shown that α₂M* promoted MMP‐9 expression and protein secretion by LRP1 in both macrophage‐derived cell lines, which was mediated by the activation of MAPK‐ERK1/2 and NF‐κB. Both intracellular signaling pathways activated by α₂M* were effectively blocked by calphostin‐C, suggesting involvement of PKC. In addition, we demonstrate that α₂M* produced extracellular calcium influx via LRP1. However, when the intracellular calcium mobilization was inhibited by BAPTA‐AM, the α₂M*‐induced MAPK‐ER1/2 activation was fully blocked in both macrophage cell lines. Finally, using specific pharmacological inhibitors for PKC, Mek1, and NF‐κB, it was shown that the α₂M*‐induced MMP‐9 protein secretion was inhibited, indicating that the MMP production promoted by the α₂M*/LRP1 interaction required the activation of both signaling pathways. These findings may prove useful in the understanding of the macrophage LRP1 role in the vascular wall during atherogenic plaque progression. J. Cell. Biochem. 111: 607–617, 2010. © 2010 Wiley‐Liss, Inc.     

Brain‐derived neurotrophic factor induces migration of endothelial cells through a TrkB–ERK–integrin αVβ3–FAK cascade

Brain‐derived neurotrophic factor (BDNF) promotes the regeneration of periodontal tissue. Since angiogenesis is important for tissue regeneration, investigating effect of BDNF on endothelial cell function may help to reveal its mechanism, whereby, BDNF promotes periodontal tissue regeneration. In this study, we examined the influence of BDNF on migration in human microvascular endothelial cells (HMVECs), focusing on the effects on extracellular signal‐regulated kinase (ERK), integrin α_Vβ₃, and focal adhesion kinase (FAK). The migration of endothelial cells was assessed with a modified Boyden chamber and a wound healing assay. The expression of integrin α_Vβ₃ and the phosphorylation of ERK and FAK were analyzed by immunoblotting and immunofluorescence microscopy. BDNF (25 ng/ml) induced cell migration. PD98059, an ERK inhibitor, K252a, a specific inhibitor for TrkB, a high affinity receptor of BDNF, and an anti‐integrin α_Vβ₃ antibody suppressed the BDNF‐induced migration. BDNF increased the levels of integrin α_Vβ₃ and phosphorylated ERK1/2 and FAK. The ERK inhibitor and TrkB inhibitor also reduced levels of integrin α_Vβ₃ and phosphorylated FAK. We propose that BDNF stimulates endothelial cell migration by a process involving TrkB/ERK/integrin α_Vβ₃/FAK, and this may help to enhance the regeneration of periodontal tissue. J. Cell. Physiol. 227: 2123–2129, 2012. © 2011 Wiley Periodicals, Inc.     

Synthesis and Biological Evaluation of Novel Oxazolo[5,4‐d]pyrimidines as Potent VEGFR‐2 Inhibitors

Tumor angiogenesis is mediated by vascular endothelial growth factor receptor (VEGFR) and other protein kinases. Inhibition of these kinases presents an attractive approach for developing anticancer therapeutics. In this work, a series of 2,5,7‐trisubstituted oxazolo[5,4‐d]pyrimidines were synthesized, and their inhibitory activities were investigated against VEGFR‐2 and human umbilical vein endothelial cells (HUVEC) in vitro. Compound 9n exhibited the most potent inhibitory activity with IC₅₀ values of 0.33 and 0.29 μM for VEGFR‐2 kinase and HUVEC, respectively. A further kinase selectivity assay revealed that these compounds exhibit good VEGFR and moderate EGFR inhibitory activities. Docking analysis suggested a common mode of interaction at the ATP‐binding site of VEGFR‐2.     

GDF‐15 is a better complimentary marker for risk stratification of arrhythmic death in non‐ischaemic,dilated cardiomyopathy than soluble ST2

Growth differentiation factor (GDF)‐15 and soluble ST2 (sST2) are established prognostic markers in acute and chronic heart failure. Assessment of these biomarkers might improve arrhythmic risk stratification of patients with non‐ischaemic, dilated cardiomyopathy (DCM) based on left ventricular ejection fraction (LVEF). We studied the prognostic value of GDF‐15 and sST2 for prediction of arrhythmic death (AD) and all‐cause mortality in patients with DCM. We prospectively enrolled 52 patients with DCM and LVEF ≤ 50%. Primary end‐points were time to AD or resuscitated cardiac arrest (RCA), and secondary end‐point was all‐cause mortality. The median follow‐up time was 7 years. A cardiac death was observed in 20 patients, where 10 patients had an AD and 2 patients had a RCA. One patient died a non‐cardiac death. GDF‐15, but not sST2, was associated with increased risk of the AD/RCA with a hazard ratio (HR) of 2.1 (95% CI = 1.1‐4.3; P = .031). GDF‐15 remained an independent predictor of AD/RCA after adjustment for LVEF with adjusted HR of 2.2 (95% CI = 1.1‐4.5; P = .028). Both GDF‐15 and sST2 were independent predictors of all‐cause mortality (adjusted HR = 2.4; 95% CI = 1.4‐4.2; P = .003 vs HR = 1.6; 95% CI = 1.05‐2.7; P = .030). In a model including GDF‐15, sST2, LVEF and NYHA functional class, only GDF‐15 was significantly associated with the secondary end‐point (adjusted HR = 2.2; 95% CI = 1.05‐5.2; P = .038). GDF‐15 is superior to sST2 in prediction of fatal arrhythmic events and all‐cause mortality in DCM. Assessment of GDF‐15 could provide additional information on top of LVEF and help identifying patients at risk of arrhythmic death.     

CD151 mediates netrin‐1‐induced angiogenesis through the Src‐FAK‐Paxillin pathway

Crosstalk between the nervous and vascular systems is important during development and in response to injury, and the laminin‐like axonal guidance protein netrin‐1 has been studied for its involvement in angiogenesis and vascular remodelling. In this study, we examined the role of netrin‐1 in angiogenesis and explored the underlying mechanisms. The effect of netrin‐1 on brain tissues and endothelial cells was examined by immunohistochemistry and western blotting in a middle cerebral artery occlusion model and in human umbilical vein endothelial cells. Cell proliferation and cell cycle progression were assessed by the MTT assay and flow cytometry, and the Transwell and tube formation assays were used to examine endothelial cell motility and function. Netrin‐1 up‐regulated CD151 and VEGF concomitant with the activation of focal adhesion kinase (FAK), Src and Paxillin in vitro and in vivo and the induction of cell proliferation, migration and tube formation in vitro. Silencing of CD151 abolished the effects of netrin‐1 on promoting cell migration and tube formation mediated by the activation of FAK/Src signalling. Netrin‐1 promoted angiogenesis in vitro and in vivo by activating the FAK/Src/Paxillin signalling pathway through a mechanism dependent on the expression of the CD151 tetraspanin, suggesting the existence of a netrin‐1/FAK/Src/CD151 signalling axis involved in the modulation of angiogenesis.     

CTGF enhances migration and MMP‐13 up‐regulation via αvβ3 integrin,FAK, ERK,and NF‐κB‐dependent pathway in human chondrosarcoma cells

Tumor malignancy is associated with several features such as proliferation ability and frequency of metastasis. Connective tissue growth factor (CTGF), a secreted protein that binds to integrins, modulates the invasive behavior of certain human cancer cells. However, the effect of CTGF on migration activity in human chondrosarcoma cells is mostly unknown. Here we found that CTGF increased the migration and expression of matrix metalloproteinase (MMP)‐13 in human chondrosarcoma cells (JJ012 cells). RGD peptide, αvβ3 monoclonal antibody (mAb) and MAPK kinase (MEK) inhibitors (PD98059 and U0126) but not RAD peptide inhibited the CTGF‐induced increase of the migration and MMP‐13 up‐regulation of chondrosarcoma cells. CTGF stimulation increased the phosphorylation of focal adhesion kinase (FAK) and extracellular signal‐regulated kinase (ERK). In addition, treatment of JJ012 cells with NF‐κB inhibitor (PDTC) or IκB protease inhibitor (TPCK) inhibited CTGF‐induced cell migration and MMP‐13 up‐regulation. Stimulation of JJ012 cells with CTGF also induced IκB kinase α/β (IKK α/β) phosphorylation, IκBα phosphorylation, p65 Ser⁵³⁶ phosphorylation, and κB‐luciferase activity. The CTGF‐mediated increases in κB‐luciferase activities were inhibited by RGD, PD98059, U0126 or FAK, and ERK2 mutant. Taken together, our results indicated that CTGF enhances the migration of chondrosarcoma cells by increasing MMP‐13 expression through the αvβ3 integrin, FAK, ERK, and NF‐κB signal transduction pathway. J. Cell. Biochem. 107: 345–356, 2009. © 2009 Wiley‐Liss, Inc.     

Macrophage inhibitory cytokine‐1 (MIC‐1/GDF15): a new marker of all‐cause mortality

Macrophage inhibitory cytokine‐1 (MIC‐1/GDF15) is a member of the TGF‐b superfamily, previously studied in cancer and inflammation. In addition to regulating body weight, MIC‐1/GDF15 may be used to predict mortality and/or disease course in cancer, cardiovascular disease (CVD), chronic renal and heart failure, as well as pulmonary embolism. These data suggested that MIC‐1/GDF15 may be a marker of all‐cause mortality. To determine whether serum MIC‐1/GDF15 estimation is a predictor of all‐cause mortality, we examined a cohort of 876 male subjects aged 35–80 years, selected from the Swedish Population Registry, and followed them for overall mortality. Serum MIC‐1/GDF15 levels were determined for all subjects from samples taken at study entry. A second (independent) cohort of 324 same‐sex twins (69% female) from the Swedish Twin Registry was similarly examined. All the twins had telomere length measured and 183 had serum levels of interleukin 6 (IL‐6) and C‐reactive protein (CRP) available. Patients were followed for up to 14 years and had cause‐specific and all‐cause mortality determined. Serum MIC‐1/GDF15 levels predicted mortality in the all‐male cohort with an adjusted odds ratio (OR) of death of 3.38 (95%CI 1.38–8.26). This finding was validated in the twin cohort. Serum MIC‐1/GDF15 remained an independent predictor of mortality when further adjusted for telomere length, IL‐6 and CRP. Additionally, serum MIC‐1/GDF15 levels were directly correlated with survival time independently of genetic background. Serum MIC‐1/GDF15 is a novel predictor of all‐cause mortality.     

Pathophysiological role of growth differentiation factor 15 (GDF15) in obesity,cancer, and cachexia

Growth differentiation factor 15 or macrophage inhibitory cytokine-1 (GDF15/MIC-1) is a divergent member of the transforming growth factor β superfamily and has a diverse pathophysiological roles in cancers, cardiometabolic disorders, and other diseases. GDF15 controls hematopoietic growth, energy homeostasis, adipose tissue metabolism, body growth, bone remodeling, and response to stress signals. The role of GDF15 in cancer development and progression is complicated and depends on the specific cancer type, stage, and tumor microenvironment. Recently, research on GDF15 and GDF15-associated signaling has accelerated due to the identification of the GDF15 receptor: glial cell line-derived neurotrophic factor (GDNF) family receptor α-like (GFRAL). Therapeutic interventions to target GDF15 and/or GFRAL revealed the mechanisms that drive its activity and might improve overall outcomes of patients with metabolic disorders and cancer. This review highlights the structure and functions of GDF15 and its receptor, emphasizing the pleiotropic role of GDF15 in obesity, tumorigenesis, metastasis, immunomodulation, and cachexia.     

Macrophage inhibitory cytokine‐1 is associated with cognitive impairment and predicts cognitive decline – the Sydney Memory and Aging Study

Higher levels of macrophage inhibitory cytokine‐1, also known as growth differentiation factor 15 (MIC‐1/GDF15), are associated with adverse health outcomes and all‐cause mortality. The aim of this study was to examine the relationships between MIC‐1/GDF15 serum levels and global cognition, five cognitive domains, and mild cognitive impairment (MCI), at baseline (Wave 1) and prospectively at 2 years (Wave 2), in nondemented participants aged 70–90 years. Analyses were controlled for age, sex, education, Framingham risk score, history of cerebrovascular accident, acute myocardial infarction, angina, cancer, depression, C‐reactive protein, tumor necrosis factor‐α, interleukins 6 and 12, and apolipoprotein ε4 genotype. Higher MIC‐1/GDF15 levels were significantly associated with lower global cognition at both waves. Cross‐sectional associations were found between MIC‐1/GDF15 and all cognitive domains in Wave 1 (all P < 0.001) and between processing speed, memory, and executive function in Wave 2 (all P < 0.001). Only a trend was found for the prospective analyses, individuals with high MIC‐1/GDF15 at baseline declined in global cognition, executive function, memory, and processing speed. However, when categorizing MIC‐1/GDF15 by tertiles, prospective analyses revealed statistically significant lower memory and executive function in Wave 2 in those in the upper tertile compared with the lower tertile. Receiver operating characteristics (ROC) analysis was used to determine MIC‐1/GDF15 cutoff values associated with cognitive decline and showed that a MIC‐1/GDF15 level exceeding 2764 pg/ml was associated with a 20% chance of decline from normal to MCI or dementia. In summary, MIC‐1/GDF15 levels are associated with cognitive performance and cognitive decline. Further research is required to determine the pathophysiology of this relationship.     

Proteomic identification of the candidate target proteins of 15‐deoxy‐delta12,14‐prostaglandin J2

15‐Deoxy‐delta12, 14‐prostaglandin J₂ (15d‐PGJ₂) is an endogenous anti‐inflammatory lipid derived from PGD₂. One potential mechanism for its activity is the covalent modification of cellular proteins, via a reactive α,β‐unsaturated carbonyl group in its cyclopentenone ring, which in turn alters protein function. In order to identify the candidate target proteins covalently modified by 15d‐PGJ₂ in human aortic endothelial cell (EC), EC was treated with biotinylated‐15d‐PGJ₂, the modified proteins extracted by Neutravidin affinity‐purification and the proteins identified by LTQ Orbitrap mass spectrometer. Classification of the 358 identified proteins was performed using PANTHER classification system ( www.pantherdb.org ), showing that the proteins mapped to metabolic process, cellular process, and transport activity. This protein data set highlights the potential for 15d‐PGJ₂ to covalently modify cellular proteins and provides a source of data that will aid further studies on the mechanism of action of this endogenous regulator of inflammation.     

生长分化因子15及其特异性受体GFRAL信号通路

生长分化因子 15(growth differentiation factor 15,GDF15)属于转化生长因子 β(transforming growth factor β,TGF-β)超家族的成员之一,是与转化生长因子β家族成员同源性很低的新一类二聚体多肽.GDF15最初发现于活化的巨噬细胞中,可通过2种不同的细...     

Growth differentiation factor 15 protects against the aging‐mediated systemic inflammatory response in humans and mice

Mitochondrial dysfunction is associated with aging‐mediated inflammatory responses, leading to metabolic deterioration, development of insulin resistance, and type 2 diabetes. Growth differentiation factor 15 (GDF15) is an important mitokine generated in response to mitochondrial stress and dysfunction; however, the implications of GDF15 to the aging process are poorly understood in mammals. In this study, we identified a link between mitochondrial stress‐induced GDF15 production and protection from tissue inflammation on aging in humans and mice. We observed an increase in serum levels and hepatic expression of GDF15 as well as pro‐inflammatory cytokines in elderly subjects. Circulating levels of cell‐free mitochondrial DNA were significantly higher in elderly subjects with elevated serum levels of GDF15. In the BXD mouse reference population, mice with metabolic impairments and shorter survival were found to exhibit higher hepatic Gdf15 expression. Mendelian randomization links reduced GDF15 expression in human blood to increased body weight and inflammation. GDF15 deficiency promotes tissue inflammation by increasing the activation of resident immune cells in metabolic organs, such as in the liver and adipose tissues of 20‐month‐old mice. Aging also results in more severe liver injury and hepatic fat deposition in Gdf15‐deficient mice. Although GDF15 is not required for Th17 cell differentiation and IL‐17 production in Th17 cells, GDF15 contributes to regulatory T‐cell‐mediated suppression of conventional T‐cell activation and inflammatory cytokines. Taken together, these data reveal that GDF15 is indispensable for attenuating aging‐mediated local and systemic inflammation, thereby maintaining glucose homeostasis and insulin sensitivity in humans and mice.     

Cross‐talk between the VEGF‐A and HGF signalling pathways in endothelial cells

Background information. Endothelial cells play a major role in angiogenesis, the process by which new blood vessels arise from a pre‐existing vascular bed. VEGF‐A (vascular endothelial growth factor‐A) is a key regulator of angiogenesis during both development and in adults. HGF (hepatocyte growth factor) is a pleiotropic cytokine that may promote VEGF‐A‐driven angiogenesis, although the signalling mechanisms underlying this co‐operation are not completely understood. Results. We analysed the effects of the combination of VEGF‐A and HGF on the activation of VEGFR‐2 (VEGF receptor‐2) and c‐met receptors, and on the stimulation of downstream signalling pathways in endothelial cells. We found that VEGFR‐2 and c‐met do not physically associate and do not transphosphorylate each other, suggesting that co‐operation involves signalling events more distal from receptor activation. We demonstrate that the VEGF isoform VEGF‐A₁₆₅ and HGF stimulate a similar set of MAPKs (mitogen‐activated protein kinases), although the kinetics and strengths of the activation differ depending on the growth factor and pathway. An enhanced activation of the signalling was observed when endothelial cells were stimulated by the combination of VEGF‐A₁₆₅ and HGF. Moreover, the combination of VEGF‐A and HGF results in a statistically significant synergistic activation of ERK1/2 (extracellular‐signal‐regulated kinase 1/2) and p38 kinases. We demonstrated that VEGF‐A₁₆₅ and HGF activate FAK (focal adhesion kinase) with different kinetics and stimulate the recruitment of phosphorylated FAK to different subsets of focal adhesions. VEGF‐A₁₆₅ and HGF regulate distinct morphogenic aspects of the cytoskeletal remodelling that are associated with the preferential activation of Rho or Rac respectively, and induce structurally distinct vascular‐like patterns in vitro in a Rho‐ or Rac‐dependent manner. Conclusions. Under angiogenic conditions, combining VEGF‐A with HGF can promote neovascularization by enhancing intracellular signalling and allowing more finely regulated control of the signalling molecules involved in the regulation of the cytoskeleton and cellular migration and morphogenesis.     

The matrix protein CCN1/CYR61 is required for αVβ5‐mediated cancer cell migration

CYR61 is one of the six proteins of the CCN family of proteins known to play diverse roles in angiogenesis, cellular proliferation, survival, migration and wound healing. However, the specific function of CYR61 in cancer is unclear, and the literature remains controversial. We used quantitative real‐time PCR to establish the expression profile of CYR61 and integrin α_Vβ₅ in three non–small cell lung cancer, five colorectal cancer, one breast cancer and one oesophageal squamous carcinoma cell lines. We showed that the levels of CYR61 were significantly increased in oesophageal squamous carcinoma cell line along with the enhanced levels of α_Vβ₅ integrin. Further, we investigated whether tumour cell–secreted CYR61 can facilitate cell migration by interacting with the α_Vβ₅ integrin. Using tumour cell lines with low, intermediate and high CYR61 expression and their isogenic variants as a cellular model, we determined that integrin α_Vβ₅ expressed on these tumour cells is required for cell migration. Moreover, we showed that the modulation of expression levels of CYR61 in these cancer cells affected their capacity for migration. These results represent an advance to the understanding of the role of CYR61 and α_vβ₅ integrin as proteins that cooperate to mediate cancer cell migration. Copyright © 2012 John Wiley & Sons, Ltd.     

CCN2/CTGF is required for matrix organization and to protect growth plate chondrocytes from cellular stress

CCN2 (connective tissue growth factor (CTGF/CCN2)) is a matricellular protein that utilizes integrins to regulate cell proliferation, migration and survival. The loss of CCN2 leads to perinatal lethality resulting from a severe chondrodysplasia. Upon closer inspection of Ccn2 mutant mice, we observed defects in extracellular matrix (ECM) organization and hypothesized that the severe chondrodysplasia caused by loss of CCN2 might be associated with defective chondrocyte survival. Ccn2 mutant growth plate chondrocytes exhibited enlarged endoplasmic reticula (ER), suggesting cellular stress. Immunofluorescence analysis confirmed elevated stress in Ccn2 mutants, with reduced stress observed in Ccn2 overexpressing transgenic mice. In vitro studies revealed that Ccn2 is a stress responsive gene in chondrocytes. The elevated stress observed in Ccn2−/− chondrocytes is direct and mediated in part through integrin α5. The expression of the survival marker NFκB and components of the autophagy pathway were decreased in Ccn2 mutant growth plates, suggesting that CCN2 may be involved in mediating chondrocyte survival. These data demonstrate that absence of a matricellular protein can result in increased cellular stress and highlight a novel protective role for CCN2 in chondrocyte survival. The severe chondrodysplasia caused by the loss of CCN2 may be due to increased chondrocyte stress and defective activation of autophagy pathways, leading to decreased cellular survival. These effects may be mediated through nuclear factor κB (NFκB) as part of a CCN2/integrin/NFκB signaling cascade.

Electronic supplementary material

The online version of this article (doi:10.1007/s12079-013-0201-y) contains supplementary material, which is available to authorized users.     

Synthesis of some novel quinone diimine derivatives of benzo‐15‐crown‐5 for application in Hg2+ recognition

A series of novel fluoroionophore bearing derivatives of benzo‐15‐crown‐5 were synthesized by the amination of benzo‐15‐crown‐5 followed by condensation with different quinones in the presence of titanium tetrachloride (TiCl₄) and 1,4‐diazabicyclo‐[2.2.2]octane. The compounds were characterized by infrared, ¹H and ¹³C nuclear magnetic resonance, mass spectroscopy and elemental analysis. Absorption and fluorescence spectral characteristics of these compounds were studied. It was observed that the anthraquinone derivative was acting as an Hg²⁺ ion sensor. Copyright © 2013 John Wiley & Sons, Ltd.     

α1‐adrenergic stimulation mediates Ca2+‐dependent inositol phosphate formation through the α1B‐like adrenoceptor subtype in adult rat cardiac myocytes

We studied the effects of increased Ca²⁺ influx on α₁‐adrenoceptor‐stimulated InsP formation in adult rat cardiac myocytes. We further examined if such effects could be mediated through a specific α₁‐adrenoceptor subtype. [³H]InsP responses to adrenaline were dependent on extracellular Ca²⁺ concentration, from 0.1 μM to 2 mM, and were completely blocked by Ca²⁺ removal. However, in cardiac myocytes preloaded with BAPTA, a highly selective calcium chelating agent, Ca²⁺ concentrations higher than 1 μM had no effect on adrenaline‐stimulated [³H]InsP formation. Taken together these results suggest that [³H]InsP formation induced by α₁‐adrenergic stimulation is in part mediated by increased Ca²⁺ influx. Consistent with this, ionomycin, a calcium ionophore, stimulated [³H]InsP formation. This response was additive with the response to adrenaline stimulation implying that different signaling mechanisms may be involved. In cardiac myocytes treated with the α_1B‐adrenoceptor alkylating agent, CEC, [³H]InsP formation remained unaffected by increased Ca²⁺ concentrations, a pattern similar to that observed when intracellular Ca²⁺ was chelated with BAPTA. In contrast, addition of the α_1A‐subtype antagonist, 5′‐methyl urapidil, did not affect the Ca²⁺ dependence of [³H]InsP formation. Neither nifedipine, a voltage‐dependent Ca²⁺ channel blocker nor the inorganic Ca²⁺ channel blockers, Ni²⁺ and Co²⁺, had any effect on adrenaline stimulated [³H]InsP, at concentrations that inhibit Ca²⁺ channels. The results suggest that in adult rat cardiac myocytes, in addition to G protein‐mediated response, α₁‐adrenergic‐stimulated [³H]InsP formation is activated by increased Ca²⁺ influx mediated by the α_1B‐subtype. J. Cell. Biochem. 84: 201–210, 2002. © 2001 Wiley‐Liss, Inc.     

Integrin expression and function in the response of primary culture hepatic stellate cells to connective tissue growth factor (CCN2)

Production of connective tissue growth factor (CCN2, also known as CTGF) is a hallmark of hepatic fibrosis. This study examined early primary cultures of hepatic stellate cells (HSC) for (i) CCN2 regulation of its cognate receptor integrin subunits; and (ii) interactions between CCN2 and integrin α₅β₁, heparan sulphate proteoglycans (HSPG) or fibronectin (FN) in supporting cell adhesion. HSC were isolated from healthy male Balb/c mice. mRNA levels of CCN2 or α₅, β₁, αv or β₃ integrin subunits were measured in days 1–7 primary culture HSC, and day 3 or day 7 cells treated with recombinant CCN2 or CCN2 small interfering RNA. Interactions between CCN2 and integrin α₅β₁, HSPG or FN were investigated using an in vitro cell adhesion assay. Co‐incident with autonomous activation over the first 7 days, primary culture HSC increasingly expressed mRNA for CCN2 or integrin subunits. Addition of exogenous CCN2 or knockdown of endogenous CCN2 differentially regulated integrin gene expression in day 3 versus day 7 cells. Either full length CCN2 (‘CCN2_1–4’) or residues 247–349 containing module 4 alone (‘CCN2₄’) supported day 3 cell adhesion in an integrin α₅β₁‐ and HSPG‐dependent fashion. Adhesion of day 3 cells to FN was promoted in an integrin α₅β₁‐dependent manner by CCN2_1–4 or CCN2₄, whereas FN promoted HSPG‐dependent HSC adhesion to CCN2_1–4 or CCN2₄. These findings suggest CCN2 regulates integrin expression in primary culture HSC and supports HSC adhesion via its binding of cell surface integrin α₅β₁, a novel CCN2 receptor in primary culture HSC which interacts co‐operatively with HSPG or FN.     

V1b vasopressin receptor trafficking and signaling: Role of arrestins,G proteins and Src kinase

The signaling pathway of G protein‐coupled receptors is strongly linked to their trafficking profile. Little is known about the molecular mechanisms involved in the vasopressin receptor V_1b subtype (V_1bR) trafficking and its impact on receptor signaling and regulation. For this purpose, we investigated the role of β‐arrestins in receptor desensitization, internalization and recycling and attempted to dissect the V_1bR‐mediated MAP kinase pathway. Using MEF cells Knocked‐out for β‐arrestins 1 and 2, we demonstrated that both β‐arrestins 1 and 2 play a fundamental role in internalization and recycling of V_1bR with a rapid and transient V_1bR‐β‐arrestin interaction in contrast to a slow and long‐lasting β‐arrestin recruitment of the V₂ vasopressin receptor subtype (V₂R). Using V_1bR‐V₂R chimeras and V_1bR C‐terminus truncations, we demonstrated the critical role of the V_1bR C‐terminus in its interaction with β‐arrestins thereby regulating the receptor internalization and recycling kinetics in a phosphorylation‐independent manner. In parallel, V_1bR MAP kinase activation was dependent on arrestins and Src‐kinase but independent on G proteins. Interestingly, Src interacted with hV_1bR at basal state and dissociated when receptor internalization occurred. Altogether, our data describe for the first time the trafficking profile and MAP kinase pathway of V_1bR involving both arrestins and Src kinase family.