CXCL9 Links Skin Inflammation and Fibrosis through CXCR3-Dependent Upregulation of Col1a1 in Fibroblasts.

Morphea is characterized by initial inflammation followed by fibrosis of the skin and soft tissue. Despite its substantial morbidity, the pathogenesis of morphea is poorly studied. Previous work showed that CXCR3 ligands CXCL9 and CXCL10 are highly upregulated in the sera and lesional skin of patients with morphea. We found that an early inflammatory subcutaneous bleomycin mouse model of dermal fibrosis mirrors the clinical, histological, and immune dysregulation observed in human morphea. We used this model to examine the role of the CXCR3 chemokine axis in the pathogenesis of cutaneous fibrosis. Using the REX3 (Reporting the Expression of CXCR3 ligands) mice, we characterized which cells produce CXCR3 ligands over time. We found that fibroblasts contribute the bulk of CXCL9-RFP and CXCL10-BFP by percentage, whereas macrophages produce high amounts on a per-cell basis. To determine whether these chemokines are mechanistically involved in pathogenesis, we treated Cxcl9-, Cxcl10-, or Cxcr3-deficient mice with bleomycin and found that fibrosis is dependent on CXCL9 and CXCR3. Addition of recombinant CXCL9 but not CXCL10 to cultured mouse fibroblasts induced Col1a1 mRNA expression, indicating that the chemokine itself contributes to fibrosis. Taken together, our studies provide evidence that CXCL9 and its receptor CXCR3 are functionally required for inflammatory fibrosis.

Kinetics and comparative reactivity of human class I and class IIb histone deacetylases.

Histone deacetylase (HDAC) enzymes modulate gene expression through the deacetylation of acetylated lysine residues on histone proteins. They operate in biological systems as part of multiprotein corepressor complexes. To understand the reactivity of isolated HDACs and the contribution of cofactor binding to reactivity, the reaction kinetics of isolated, recombinant human HDACs 1, 2, 3, 6, 8, and 10 were measured using a novel, continuous protease-coupled enzyme assay. Values of k(cat) and k(cat)/K(m) and the pH dependence of these values were determined for the reactions of each isozyme with acetyl-Gly-Ala-(N(epsilon)-acetyl-Lys)-AMC. Values of k(cat) spanned the range of 0.006-2.8 s(-1), and k(cat)/K(m) values ranged from 60 to 110000 M(-1) s(-1). The pH profiles for both k(cat) and k(cat)/K(m) were bell-shaped for all of the HDAC isozymes, with pH optima at approximately pH 8. Values of K(i) for the inhibitor trichostatin A were determined for each isozyme. The inhibition constants were generally similar for all HDAC isozymes, except that the value for HDAC8 was significantly higher than that for the other isozymes. The reaction of HDAC8 with an alternative substrate was performed to assess the steric requirements of the HDAC8 active site, and the effect of phosphorylation on HDAC1 activity was examined. The results are discussed in terms of the biological roles of the HDAC enzymes and the proposed reaction mechanism of acetyllysine hydrolysis by these enzymes.