John Crean

Matrix metalloproteinases in the human intervertebral disc: role in disc degeneration and scoliosis.

Study Design. Biochemical study of human intervertebral discs collected at surgery from patients with low back pain associated with disc degeneration or scoliosis. Matrix metalloproteinases were studied by quantitative zymography. Objective. To determine whether changes in the expression of matrix metalloproteinases will bring about tissue remodelling that contributes to the progressive nature and pathology of these diseases of the intervertebral disc. Summary of Background Data. The diseases of the intervertebral disc, degenerative disc disease and scoliosis, are both characterized by changes in the extracellular matrix components that will affect the mechanical function of the tissue. Matrix metalloproteinases are known to have the capability of degrading all the known extracellular matrix components of the disc. Methods. Matrix metalloproteinases 2 and 9 were detected by gelatin‐gel zymography and quantified by laser scanning densitometry. Both pro and active forms of the enzymes were measured. Thirty‐four discs from patients with low back pain and 29 from patients with scoliosis were investigated. Results. A correlation was found between the increasing levels of matrix metalloproteinases 2 and 9 and the grade of degenerative disc disease. In addition, the levels of these enzymes show a differential expression across the scoliotic disc with the highest levels in samples taken from the convexity of the curve. Conclusions. The difference between the concave and convex side of the scoliotic curve indicates that mechanical loads might influence the expression of these enzymes. The increased expression of these enzymes in both degenerative disc disease and scoliosis strongly suggests that they may affect the progressive nature of these diseases.

Changes in collagen cross-linking in degenerative disc disease and scoliosis

Study Design. Biochemical study of the changes in the collagen cross‐link profile of human intervertebral discs collected at surgery from patients with either low back pain associated with disc degeneration or scoliosis. Objective. To determine whether changes occur in the collagen cross‐link profile in the disc of patients with either low back pain associated with disc degeneration or scoliosis, which may well influence matrix integrity. Such changes in the cross‐link profile of a tissue indicates increased matrix turnover and tissue remodeling and may have implications for the progression of these disorders. Summary of Background Data. The diseases of the intervertebral disc, degenerative disc disease and scoliosis, are both characterized by changes in the extracellular matrix components that will affect the mechanical function of the tissue. The stability of the collagenous components and hence the mechanical integrity of connective tissues such as the disc is dependent on the degree and type of cross‐links between the collagen molecules. This article reports results on the distribution of the different cross‐links in the disc and the changes that occur with age, degenerative disc disease, and scoliosis. Methods. Thirty‐three discs were obtained from patients with degenerative disc disease and 29 discs from patients with scoliosis. Samples were acid hydrolyzed and the collagen cross‐links analyzed by either fractionation on an amino acid analyzer configured for cross‐link analysis using ninhydrin postcolumn detection or fractionation by high‐pressure liquid chromatography with fluorescence detection. Results. The reducible cross‐links and the mature cross‐link all increased from the outer anulus fibrosus through into the nucleus pulposus. The highest levels of the mature cross‐link were found in the cartilage endplate. The nonenzymic derived cross‐link, pentosidine, in contrast, showed little difference across the disc, but did show the expected age‐related increase. In degenerative disc disease, no change in the levels of the reducible or mature cross‐links was found, but a decrease was observed in the levels of the age‐related cross‐link pentosidine in the more severe disease samples. In scoliosis, significantly higher levels of the reducible cross‐links were found on the convex than on the concave side of the scoliotic disc. Conclusions. These changes in the cross‐link profile of the intervertebral disc in degenerative disc disease and scoliosis are indicative of increased matrix turnover and tissue remodeling and likely to have implications for the progression of these disorders.

Elevated aqueous humour tissue inhibitor of matrix metalloproteinase-1 and connective tissue growth factor in pseudoexfoliation syndrome

Background/aims: Pseudoexfoliation syndrome (PXF) was recently found to be associated with increased expression of transforming growth factor β1 (TGFβ1) in the aqueous humour. As concern has been raised regarding anti-TGFβ therapy, which can potentially disrupt the maintenance of anterior chamber asso-ciated immune deviation, the authors explored the levels of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), matrix metalloproteinase-9 (MMP-9), and connective tissue growth factor (CTGF) in aqueous humour to determine if these may represent alternative therapeutic targets. Methods: Aqueous humour samples were collected from patients who underwent routine cataract surgery. All patients were categorised into three main groups—PXF, uveitis, and control. The PXF group was further subcategorised into three grades based on the density of the exfoliative material observed on biomicroscopy, as well as the presence or absence of glaucoma. TIMP-1, MMP-9, and CTGF levels were measured using specific enzyme immunoassays (ELISA). Results: Eyes with PXF had significantly higher aqueous humour TIMP-1 concentration (n = 56, mean (SE), 9.76 (1.10) ng/ml) compared with controls (n = 112, 5.73 (0.43) ng/ml, p<0.01). Similarly, the CTGF level in PXF eyes (n = 36, 4.38 (0.65) ng/ml) was higher than controls (n = 29, 2.35 (0.46) ng/ml, p<0.05). Further, the CTGF concentration in the PXF glaucoma group is significantly higher compared with PXF eyes without glaucoma (6.03 (1.09) ng/ml v 2.73 (0.45) ng/ml, p<0.01). The MMP-9 levels were low and below detection limit in all PXF and control samples with no statistical difference between groups. Conclusion: A raised TIMP-1 level and a low MMP-9 level in aqueous humour of PXF eyes may imply a downregulation in proteolytic activity. The increased CTGF concentration supports the proposed fibrotic pathology of PXF. Regulation of MMP/TIMP expression and anti-CTGF therapy may offer potential therapeutic avenues for controlling PXF associated ocular morbidity.

Lipoxin A4 Redistributes Myosin IIA and Cdc42 in Macrophages: Implications for Phagocytosis of Apoptotic Leukocytes

Lipoxins (LXs) are endogenously produced anti-inflammatory agents that modulate leukocyte trafficking and stimulate nonphlogistic macrophage phagocytosis of apoptotic neutrophils, thereby promoting the resolution of inflammation. Previous data suggest a role for altered protein phosphorylation and cytoskeletal rearrangement in LX-stimulated phagocytosis but the exact mechanisms remain unclear. In this study we examine the effects of LXA4 on the protein phosphorylation pattern of THP-1 cells differentiated into a macrophage-like phenotype. THP-1 cells stimulated with LXA4 (1 nM) exhibit dephosphorylation of a 220-kDa protein. Using mass spectrometry, this protein was identified as MYH9, a nonmuscle myosin H chain II isoform A, which is involved in cytoskeleton rearrangement. THP-1 cells treated with LXA4 adopt a polarized morphology with activated Cdc42 localized toward the leading edge and MYH9 localized at the cell posterior. Polarized distribution of Cdc42 is associated with Akt/PKB-mediated Cdc42 activation. Interestingly, the annexin-derived peptide Ac2–26, a recently described agonist for the LXA4 receptor, also stimulates macrophage phagocytosis, MYH9 dephosphorylation, and MYH9 redistribution. In addition, we demonstrate that LXA4 stimulates the phosphorylation of key polarity organization molecules: Akt, protein kinase Cζ, and glycogen synthase kinase-3β. Inhibition of LXA4-induced Akt and protein kinase Cζ activity with specific inhibitors prevented LXA4-stimulated phagocytosis of both apoptotic polymorphonuclear neutrophils and lymphocytes, highlighting a potential use for LXA4 in the treatment of autoimmune diseases. Furthermore, phosphorylation and subsequent inactivation of glycogen synthase kinase-3β resulted in an increase in phagocytosis similar to that of LXA4. These data highlight an integrated mechanism whereby LXA4 regulates phagocytosis through facilitative actin cytoskeleton rearrangement and cell polarization.

Connective tissue growth factor [CTGF]/CCN2 stimulates mesangial cell migration through integrated dissolution of focal adhesion complexes and activation of cell polarization

Connective tissue growth factor [CTGF]/CCN2 is a prototypic member of the CCN family of regulatory proteins. CTGF expression is up‐regulated in a number of fibrotic diseases, including diabetic nephropathy, where it is believed to act as a downstream mediator of TGF‐β function; however, the exact mechanisms whereby CTGF mediates its effects remain unclear. Here, we describe the role of CTGF in cell migration and actin disassembly in human mesangial cells, a primary target in the development of renal glomerulosclerosis. The addition of CTGF to primary mesangial cells induced cell migration and cytoskeletal rearrangement but had no effect on cell proliferation. Cytoskeletal rearrangement was associated with a loss of focal adhesions, involving tyrosine dephosphorylation of focal adhesion kinase and paxillin, increased activity of the protein tyrosine phosphatase SHP‐2, with a concomitant decrease in RhoA and Rac1 activity. Conversely, Cdc42 activity was increased by CTGF. These functional responses were associated with the phosphorylation and translocation of protein kinase C‐ζ to the leading edge of migrating cells. Inhibition of CTGF‐induced protein kinase C‐ζ activity with a myristolated PKC‐ζ inhibitor prevented cell migration. Moreover, transient transfection of human mesangial cells with a PKC‐ζ kinase inactive mutant (dominant negative) expression vector also led to a decrease in CTGF‐induced migration compared with wild‐type. Furthermore, CTGF stimulated phosphorylation and activation of GSK‐3β. These data highlight for the first time an integrated mechanism whereby CTGF regulates cell migration through facilitative actin cytoskeleton disassembly, which is mediated by dephosphorylation of focal adhesion kinase and paxillin, loss of RhoA activity, activation of Cdc42, and phosphorylation of PKC‐ζ and GSK‐3β. These changes indicate that the initial stages of CTGF mediated mesangial cell migration are similar to those involved in the process of cell polarization. These findings begin to shed mechanistic light on the renal diabetic milieu, where increased CTGF expression in the glomerulus contributes to cellular dysfunction.

Connective Tissue Growth Factor Is Increased in Pseudoexfoliation Glaucoma

PURPOSE Pseudoexfoliation (PXF) syndrome is a generalized disorder of the extracellular matrix (ECM) involving the trabecular meshwork (TM), associated with raised intraocular pressure, glaucoma, and cataract. The purposes of this study were to quantify aqueous humor connective tissue growth factor (CTGF) in PXF glaucoma, to determine the effect of CTGF on ECM production in TM cells, and to identify intracellular CTGF signaling pathways. METHODS Aqueous humor samples were obtained from patients undergoing routine cataract surgery or trabeculectomy. CTGF levels were quantified by ELISA. The effect of CTGF on fibrillin-1 expression in TM cells was investigated by real-time PCR. Western immunoblot analysis was used to investigate CTGF signaling. c-Jun/AP-1 activation was measured in CHO cells by ELISA after stimulation with CTGF. RESULTS PXF with glaucoma had the highest aqueous humor level of CTGF (n = 18; 5.15 ± 0.79 ng/mL [SEM]; P < 0.01) compared with PXF without glaucoma (n = 15; 2.76 ± 0.64 ng/mL), primary open-angle glaucoma (POAG; n = 20; 3.05 ± 0.40 ng/mL), and the control (n = 21; 2.60 ± 0.29 ng/mL). In vitro exposure of TM cells to CTGF resulted in a 50% upregulation of fibrillin-1, which was partially blocked with the MEK (mitogen-activated protein extracellular kinase) inhibitor PD098059. Western blot analysis demonstrated increased phosphorylation of p42/44 MAPK, p38 MAPK, and the JNK pathways in response to CTGF. c-Jun/AP-1 activity was significantly increased in response to CTGF treatment. CONCLUSIONS Increased levels of CTGF in the aqueous humor of PXF patients likely has pathologic significance through increased production of fibrillin-1 by TM cells through activation of p42/44 MAPK, p38 MAPK, and JNK pathways.

CTGF/CCN2 activates canonical Wnt signalling in mesangial cells through LRP6: implications for the pathogenesis of diabetic nephropathy.

We describe the activation of Wnt signalling in mesangial cells by CCN2. CCN2 stimulates phosphorylation of LRP6 and GSK‐3β resulting in accumulation and nuclear localisation of β‐catenin, TCF/LEF activity and expression of Wnt targets. This is coincident with decreased phosphorylation of β‐catenin on Ser 33/37 and increased phosphorylation on Tyr142. DKK‐1 and LRP6 siRNA reversed CCN2s effects. Microarray analyses of diabetic patients identified differentially expressed Wnt components. β‐Catenin is increased in type 1 diabetic and UUO mice and in in vitro models of hyperglycaemia and hypertension. These findings suggest that Wnt/CCN2 signalling plays a role in the pathogenesis of diabetic nephropathy.

Jagged/Notch signalling is required for a subset of TGFβ1 responses in human kidney epithelial cells.

The Jagged/Notch pathway has been implicated in TGFβ1 responses in epithelial cells in diabetic nephropathy and other fibrotic conditions in vivo. Here, we identify that Jagged/Notch signalling is required for a subset of TGFβ1-stimulated gene responses in human kidney epithelial cells in vitro. TGFβ1 treatment of HK-2 and RPTEC cells for 24h increased Jagged1 (a Notch ligand) and Hes1 (a Notch target) mRNA. This response was inhibited by co-incubation with Compound E, an inhibitor of γ-secretase (GSI), an enzyme required for Notch receptor cleavage and transcription regulation. In both cell types, TGFβ1-responsive genes associated with epithelial-mesenchymal transition such as E-cadherin and vimentin were also affected by γ-secretase inhibition, but other TGFβ1 targets such as connective tissue growth factor (CTGF) and thrombospondin-1 (THBS1) were not. TGFβ1-induced changes in Jagged1 expression preceded EMT-associated gene changes, and co-incubation with GSI altered TGFβ1-induced changes in cell shape and cytoskeleton. Transfection of cells with the activated, cleaved form of Notch (NICD) triggered decreased expression of E-cadherin in the absence of TGFβ1, but did not affect α-smooth muscle actin expression, suggesting differential requirements for Notch signalling within the TGFβ1-responsive gene subset. Increased Jagged1 expression upon TGFβ1 exposure required Smad3 signalling, and was also regulated by PI3K and ERK. These data suggest that Jagged/Notch signalling is required for a subset of TGFβ1-responsive genes, and that complex signalling pathways are involved in the crosstalk between TGFβ1 and Notch cascades in kidney epithelia.

Connective tissue growth factor/CCN2 stimulates actin disassembly through Akt/protein kinase B-mediated phosphorylation and cytoplasmic translocation of p27Kip-1

Connective tissue growth factor (CTGF/ CCN2) is a 38‐kDa secreted protein, a prototypic member of the CCN family, which is up‐regulated in many diseases, including atherosclerosis, pulmonary fibrosis, and diabetic nephropathy. We previously showed that CTGF can cause actin disassembly with concurrent down‐regulation of the small GTPase Rho A and proposed an integrated signaling network connecting focal adhesion dissolution and actin disassembly with cell polarization and migration. Here, we further delineate the role of CTGF in cell migration and actin disassembly in human mesangial cells, a primary target in the development of renal glomerulosclerosis. The functional response of mesangial cells to treatment with CTGF was associated with the phosphorylation of Akt/ protein kinase B (PKB) and resultant phosphorylation of a number of Akt/PKB substrates. Two of these substrates were identified as FKHR and p27Kip‐1. CTGF stimulated the phosphorylation and cytoplasmic translocation of p27Kip‐1 on serine 10. Addition of the PI‐3 kinase inhibitor LY294002 abrogated this response; moreover, addition of the Akt/PKB inhibitor interleu‐kin (IL)‐6‐hydroxymethyl‐chiro‐inositol‐2(R)‐2‐methyl‐3‐O‐octadecylcarbonate prevented p27Kip‐1 phosphorylation in response to CTGF. Immunocytochemistry revealed that serine 10 phosphorylated p27Kip‐1 colocalized with the ends of actin filaments in cells treated with CTGF. Further investigation of other Akt/PKB sites on p27Kip‐1, revealed that phosphorylation on threonine 157 was necessary for CTGF mediated p27Kip‐1 cyto‐plasmic localization; mutation of the threonine 157 site prevented cytoplasmic localization, protected against actin disassembly and inhibited cell migration. CTGF also stimulated an increased association between Rho A and p27Kip‐1. Interestingly, this resulted in an increase in phosphorylation of LIM kinase and subsequent phosphorylation of cofilin, suggesting that CTGF mediated p27Kip‐1 activation results in uncoupling of the Rho A/LIM kinase/cofilin pathway. Confirming the central role of Akt/PKB, CTGF‐stimulated actin depolymerization only in wild‐type mouse embryonic fibroblasts (MEFs) compared to Akt‐1/3 (PKB α/γ) knockout MEFs. These data reveal important mechanistic insights into how CTGF may contribute to mesangial cell dysfunction in the diabetic milieu and sheds new light on the proposed role of p27Kip‐1 as a mediator of actin rearrangement.—Crean, J. K., Furlong, F., Mitchell, D., McArdle E., Godson, C., and Martin, F. Connective tissue growth factor/CCN2 stimulates actin disassembly through Akt/protein kinase B‐mediated phosphorylation and cytoplasmic translocation of p27Kip‐1. FASEB J. 20, E1037‐E1048 (2006)

Connective tissue growth factor: an attractive therapeutic target in fibrotic renal disease

Despite diverse initiating insults, glomerulosclerosis and tubulointerstitial fibrosis are pathological features common to most forms of progressive renal disease. Control of systemic hypertension and blockade of the renin-angiotensin system ameliorate the rate of progression of chronic renal disease; however they generally fail to completely arrest the scarring process. While the chain of events leading to scarring are still being defined, TGF-β is a cytokine that plays a pivotal role in the pathogenesis of glomerulosclerosis and tubulointerstitial fibrosis [1]. Given the pleiotropic effects of TGF-β, significant attention has focused on the potential of its downstream mediators as therapeutic targets. Connective tissue growth factor (CTGF) is a member of the CCN gene family, which includes CyR61 (cysteine rich 61), Nov (Nephroblastoma overexpressed) and the WISP family (for review see [2,3,4]). These immediate-early genes coordinate complex biologic processes during differentiation and tissue repair [5]. Increased expression of CTGF has been detected in experimental and human renal fibrosis where it correlates with glomerulosclerosis and the degree of tubulointerstitial fibrosis [6]. In these settings CTGF expression is regulated at least in part by TGF-β. This review details the biology of CTGF with specific reference to its potential as a therapeutic target in renal fibrosis.