BackgroundConnective tissue growth factor (CTGF) is widely thought to promote the development of fibrosis in collaboration with transforming growth factor (TGF)-β; however, most of the evidence for its involvement comes from correlative and culture-based studies. In this study, the importance of CTGF in tissue fibrosis was directly examined in three murine models of fibrotic disease: a novel model of multiorgan fibrosis induced by repeated intraperitoneal injections of CTGF and TGF-β2; the unilateral ureteral obstruction (UUO) renal fibrosis model; and an intratracheal bleomycin instillation model of pulmonary fibrosis.ResultsIntraperitoneal coadministration of CTGF and TGF-β2 elicited a profound fibrotic response that was inhibited by the human anti-CTGF antibody FG-3019, as indicated by the ability of FG-3019 to ameliorate the histologic signs of fibrosis and reduce the otherwise increased hydroxyproline:proline (Hyp:Pro) ratios by 25% in kidney (P < 0.05), 30% in liver (P < 0.01) and 63% in lung (P < 0.05). Moreover, administration of either cytokine alone failed to elicit a fibrotic response, thus demonstrating that CTGF is both necessary and sufficient to initiate fibrosis in the presence of TGF-β and vice versa. In keeping with this requirement for CTGF function in fibrosis, FG-3019 also reduced the renal Hyp:Pro response up to 20% after UUO (P < 0.05). In bleomycin-injured animals, a similar trend towards a FG-3019 treatment effect was observed (38% reduction in total lung Hyp, P = 0.056). Thus, FG-3019 antibody treatment consistently reduced excessive collagen deposition and the pathologic severity of fibrosis in all models.ConclusionCooperative interactions between CTGF and TGF-β signaling are required to elicit overt tissue fibrosis. This interdependence and the observed anti-fibrotic effects of FG-3019 indicate that anti-CTGF therapy may provide therapeutic benefit in different forms of fibroproliferative disease.

Hypoxia-inducible transcription factor (HIF) is regulated by two oxygen-dependent events that are catalyzed by the HIF prolyl 4-hydroxylases (HIF-P4Hs) and HIF asparaginyl hydroxylase (FIH). We have purified the three recombinant human HIF-P4Hs to near homogeneity and characterized their catalytic properties and inhibition and those of FIH. The specific activities of the HIF-P4Hs were at least 40-50 mol/mol/min, and they and FIH catalyzed an uncoupled decarboxylation of 2-oxoglutarate in the absence of any peptide substrate. The purified HIF-P4Hs showed considerable activities even without added Fe2+, their apparent Km values for iron being markedly lower than that of FIH. Desferrioxamine and several metals were effective inhibitors of FIH, but surprisingly, ineffective inhibitors of the HIF-P4Hs in vitro, especially of HIF-P4H-2. Desferrioxamine and cobalt were more effective in cultured insect cells synthesizing recombinant HIF-P4H-2, but complete inhibition was not achieved and most of the enzyme was inactivated irreversibly. Cobalt also rapidly inactivated HIF-P4Hs during storage at 4 degrees C. The well-known stabilization of HIF-alpha by cobalt and nickel is thus not due to a simple competitive inhibition of HIF-P4Hs. The effective inhibition of FIH by these metals and zinc probably leads to full transcriptional activity of HIF-alpha even in concentrations that produce no stabilization of HIF-alpha.

In diabetic nephropathy, connective tissue growth factor (CTGF) is upregulated and bone morphogenetic protein 7 (BMP-7) is downregulated. CTGF is known to inhibit BMP-4, but similar cross-talk between BMP-7 and CTGF has not been studied. In this study, it was hypothesized that CTGF acts as an inhibitor of BMP-7 signaling activity in diabetic nephropathy. Compared with diabetic wild-type CTGF ϩ/ϩ mice, diabetic CTGF ϩ/Ϫ mice had approximately 50% lower CTGF mRNA and protein, less severe albuminuria, no thickening of the glomerular basement membrane, and preserved matrix metalloproteinase (MMP) activity. Although the amount of BMP-7 mRNA was similar in the kidneys of diabetic CTGF ϩ/ϩ and CTGF ϩ/Ϫ mice, phosphorylation of the BMP signal transduction protein Smad1/5 and expression of the BMP target gene Id1 were lower in diabetic CTGF ϩ/ϩ mice.Moreover, renal Id1 mRNA expression correlated with albuminuria (R ϭ Ϫ0.86) and MMP activity (R ϭ 0.76). In normoglycemic mice, intraperitoneal injection of CTGF led to a decrease of pSmad1/5 in the renal cortex. In cultured renal glomerular and tubulointerstitial cells, CTGF diminished BMP-7 signaling activity, evidenced by lower levels of pSmad1/5, Id1 mRNA, and BMP-responsive elementluciferase activity. Co-immunoprecipitation, solid-phase binding assay, and surface plasmon resonance analysis showed that CTGF binds BMP-7 with high affinity (Kd approximately 14 nM). In conclusion, upregulation of CTGF inhibits BMP-7 signal transduction in the diabetic kidney and contributes to altered gene transcription, reduced MMP activity, glomerular basement membrane thickening, and albuminuria, all of which are hallmarks of diabetic nephropathy.

Connective tissue growth factor (CTGF) plays a key role in renal fibrosis. Urinary CTGF is elevated in various renal diseases and may have biomarker potential. However, it is unknown which processes contribute to elevated urinary CTGF levels. Thus far, urinary CTGF was considered to reflect renal expression. We investigated how tubular dysfunction affects urinary CTGF levels. To study this, we administered recombinant CTGF intravenously to rodents. We used both full-length CTGF and the NH(2)-terminal fragment, since the NH(2)-fragment is the predominant form detected in urine. Renal CTGF extraction, determined by simultaneous arterial and renal vein sampling, was 18 +/- 3% for full-length CTGF and 21 +/- 1% for the NH(2)-fragment. Fractional excretion was very low for both CTGFs (0.02 +/- 0.006% and 0.10 +/- 0.02%, respectively), indicating that >99% of the extracted CTGF was metabolized by the kidney. Immunohistochemistry revealed extensive proximal tubular uptake of CTGF in apical endocytic vesicles and colocalization with megalin. Urinary CTGF was elevated in megalin- and cubilin-deficient mice but not in cubilin-deficient mice. Inhibition of tubular reabsorption by Gelofusine reduced renal uptake of CTGF and increased urinary CTGF. In healthy volunteers, Gelofusine also induced an increase of urinary CTGF excretion, comparable to the increase of beta(2)-microglobulin excretion (r = 0.99). Furthermore, urinary CTGF correlated with beta(2)-microglobulin (r = 0.85) in renal disease patients (n = 108), and only beta(2)-microglobulin emerged as an independent determinant of urinary CTGF. Thus filtered CTGF is normally reabsorbed almost completely in proximal tubules via megalin, and elevated urinary CTGF may largely reflect proximal tubular dysfunction.

Three human prolyl 4-hydroxylases (P4Hs) regulate the hypoxia-inducible transcription factors (HIFs) by hydroxylating a Leu-Xaa-Xaa-Leu-Ala-Pro motif. We report here that the two leucines in the Leu-Glu-Met-LeuAla-Pro core motif of a 20-residue peptide corresponding to the sequence around Pro 564 in HIF-1␣ can be replaced by many residues with no or only a modest decrease in its substrate properties or in some cases even a slight increase. The glutamate and methionine could be substituted by almost any residue, eight amino acids in the former position and four in the latter being even better for HIF-P4H-3 than the wild-type residues. Alanine was by far the strictest requirement, because no residue could fully substitute for it in the case of HIF-P4H-1, and only serine or isoleucine, valine, and serine did this in the cases of HIF-P4Hs 2 and 3. Peptides with more than one substitution, having the core sequences Trp-Glu-Met-Val-Ala-Pro, Tyr-Glu-Met-Ile-Ala-Pro, IleGlu-Met-Ile-Ala-Pro, Trp-Glu-Met-Val-Ser-Pro, and TrpGlu-Ala-Val-Ser-Pro were in most cases equally as good or almost as good substrates as the wild-type peptide. The acidic residues present in the 20-residue peptide also played a distinct role, but alanine substitution for any six of them, and in some combinations even three of them, had no negative effects. Substitution of the proline by 3,4-dehydroproline or L-azetidine-2-carboxylic acid, but not any other residue, led to a high rate of uncoupled 2-oxoglutarate decarboxylation with no hydroxylation. The data obtained for the three HIF-P4Hs in various experiments were in most cases similar, but in some cases HIF-P4H-3 showed distinctly different properties.The hypoxia-inducible transcription factors (HIFs), 1 which are essential for the regulation of cellular and systemic oxygen homeostasis, are ␣␤ heterodimers in which both types of subunit are basic helix-loop-helix Per-Arnt-Sim proteins. The human ␣ subunit has three isoforms, HIF-1␣ to HIF-3␣, of which HIF-1␣ and HIF-2␣ are expressed constitutively but are rapidly degraded under normoxic conditions (for reviews see Refs.