William T. Roswit

Integrin α2β1 (VLA-2) mediates reorganization and contraction of collagen matrices by human cells

The capacity of cells to organize and contract collagen fibrils is fundamental to processes as diverse as embryogenesis and wound healing. We analyzed different beta 1 integrins on diploid fibroblasts for their role in modifying the tertiary structure of collagen matrices. Using monoclonal antibodies that block the interaction of integrins with their ligands, evidence was obtained that alpha 2 beta 1 integrin is required for the contraction of a type I collagen matrix. Further supporting the role of alpha 2 beta 1, cell lines expressing minimal levels of this integrin uniformly failed to contract collagen matrices. In addition, transfection of a full-length alpha 2 cDNA into one such cell line led to enhanced cell surface expression of alpha 2 beta 1 and conferred the de novo capacity to contract collagen matrices.

Blocking airway mucous cell metaplasia by inhibiting EGFR antiapoptosis and IL-13 transdifferentiation signals

Epithelial hyperplasia and metaplasia are common features of inflammatory and neoplastic disease, but the basis for the altered epithelial phenotype is often uncertain. Here we show that long-term ciliated cell hyperplasia coincides with mucous (goblet) cell metaplasia after respiratory viral clearance in mouse airways. This chronic switch in epithelial behavior exhibits genetic susceptibility and depends on persistent activation of EGFR signaling to PI3K that prevents apoptosis of ciliated cells and on IL-13 signaling that promotes transdifferentiation of ciliated to goblet cells. Thus, EGFR blockade (using an irreversible EGFR kinase inhibitor designated EKB-569) prevents virus-induced increases in ciliated and goblet cells whereas IL-13 blockade (using s-IL-13Ralpha2-Fc) exacerbates ciliated cell hyperplasia but still inhibits goblet cell metaplasia. The distinct effects of EGFR and IL-13 inhibitors after viral reprogramming suggest that these combined therapeutic strategies may also correct epithelial architecture in the setting of airway inflammatory disorders characterized by a similar pattern of chronic EGFR activation, IL-13 expression, and ciliated-to-goblet cell metaplasia.

Direct Suppression of Stat1 Function during Adenoviral Infection

The action of adenoviral E1A oncoprotein on host immune-response genes has been attributed to interaction with p300/CBP-type transcriptional coactivators in competition with endogenous transcription factors such as signal transducer and activator of transcription (STAT) proteins. However, we show that mutant forms of E1A that no longer bind p300/CBP can still interact directly with Stat1 (via E1A N-terminal and Stat1 C-terminal residues) and block IFNgamma-driven, Stat1-dependent gene activation and consequent function during early-phase infection in the natural host cell. The results provide a distinct and more specific mechanism for E1A-mediated immune suppression and an alternative model of IFNgamma-driven enhanceosome formation that may allow for other adaptors (in addition to p300/CBP) to link Stat1 to the basal transcription complex.

Human Skin Fibroblast Collagenase: Interaction with Substrate and Inhibitor

Human skin fibroblasts secrete collagen, procollagenase and a collagenase inhibitor. This study addresses the nature of the interaction between these important fibroblast products. The binding of procollagenase and of active collagenase to native collagen in solution was examined by employing Sephadex G-150 gel-filtration chromatography to separate bound versus unbound enzyme. Active enzyme bound readily to collagen; the equilibrium constant of binding, Kd, was calculated to be 5.1 to 10(-7)M. Thus, collagenase binds with nearly equal affinity to both monomeric collagen and aggregated fibrils (Kd = 9 X 10(-7)M; [Welgus et al., 1980]). Furthermore, since Kd congruent to Km congruent to 10(-6)M, the ratio k2/k1 must be extremely small, directly implicating the catalytic step represented by the rate constant k2, and not the binding of enzyme to substrate, as the rate-limiting step of collagenase action. In contrast, procollagenase demonstrated no capacity to bind to collagen. The interaction of procollagenase and of active collagenase with inhibitor was examined utilizing both conventional and high-precision liquid gel-filtration chromatography. A higher molecular weight complex could be demonstrated consisting of active collagenase and inhibitor; no such interaction occurred between procollagenase and the inhibitory protein. Analysis of Lineweaver-Burk plots showed that inhibition was accompanied by a corresponding change in Vmax; Km remained unchanged. Such results are indicative of a noncompetitive mechanism of inhibition and are consistent with the formation of an enzyme-inhibitor complex. The Ki of enzyme-inhibitor binding was determined to be less than 10(-9)M. The data indicate that procollagenase can neither interact with its specific inhibitor nor bind to collagen. Extracellular activation of the collagenase zymogen is thus a critical event, which can be followed either by binding to substrate or interaction with inhibitor.

Targeted inhibition of interferon-gamma-dependent intercellular adhesion molecule-1 (ICAM-1) expression using dominant-negative Stat1.

A subset of epithelial immune-response genes (including intercellular adhesion molecule-1 (ICAM-1)) depends on an IFN-γ signal transduction pathway with the Stat1 transcription factor as a critical intermediate. Excessive local activation of this pathway may lead to airway inflammation, so we sought to selectively down-regulate the pathway using a dominant-negative strategy for inhibition of epithelial Stat1 in a primary culture airway epithelial cell model. Using a Stat1-deficient cell line, we demonstrated that transfection of wild-type Stat1 expression plasmid restored appropriate Stat1 expression and IFN-γ-dependent phosphorylation as well as consequent IFN-γ activation of cotransfected ICAM-1 promoter constructs and endogenous ICAM-1 gene expression. However, mutations of Stat1 at Tyr-701 (JAK kinase phosphorylation site), Glu-428/429 (putative DNA-binding site), His-713 (splice site resulting in Stat1β formation), or Ser-727 (MAP kinase phosphorylation site) all decreased Stat1 capacity to activate the ICAM-1 promoter. The Tyr-701 mutant (followed by the His-713 mutant) were most effective in disabling Stat1 function and in overcoming the activating effect of cotransfected wild-type Stat1 in this cell system thereby highlighting the effectiveness of blocking Stat1 homo- and hetero-dimerization. In experiments using primary culture human tracheobronchial epithelial cells (hTBECs) and each of the four Stat1 mutant plasmids, transfection with the Tyr-701 and His-713 mutants again most effectively inhibited IFN-γ activation of an ICAM-1 gene promoter construct. Then by transfecting hTBECs with wild-type or mutant Stat1 tagged with a Flag reporter sequence, we used dual immunofluorescence to show that hTBECs expressing the Tyr-701 or His-713 mutants were prevented from expressing endogenous ICAM-1 in response to IFN-γ treatment. The capacity of a specific Stat1 mutations to exert a potent dominant-negative effect on IFN-γ signal transduction provides for further definition of Stat1 structure function and a means for natural or engineered expression of mutant Stat1 to selectively down-regulate activity of this pathway in a cell type- or tissue-specific manner during immune and/or inflammatory responses.

IL-13–induced airway mucus production is attenuated by MAPK13 inhibition

Increased mucus production is a common cause of morbidity and mortality in inflammatory airway diseases, including asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis. However, the precise molecular mechanisms for pathogenic mucus production are largely undetermined. Accordingly, there are no specific and effective anti-mucus therapeutics. Here, we define a signaling pathway from chloride channel calcium-activated 1 (CLCA1) to MAPK13 that is responsible for IL-13-driven mucus production in human airway epithelial cells. The same pathway was also highly activated in the lungs of humans with excess mucus production due to COPD. We further validated the pathway by using structure-based drug design to develop a series of novel MAPK13 inhibitors with nanomolar potency that effectively reduced mucus production in human airway epithelial cells. These results uncover and validate a new pathway for regulating mucus production as well as a corresponding therapeutic approach to mucus overproduction in inflammatory airway diseases.

Purification and properties of rat uterine procollagenase

A procollagenase from monolayer cultures of postpartum rat uterine cells has been purified. The crucial step in the purification is the binding of the procollagenase from crude, fetal bovine serum-containing culture medium to heparin-Sepharose, followed by elution with extremely low concentrations (5-10 nM) of dextran sulfate. Resultant eluates contain 8-10% procollagenase. Purification is completed by ion-exchange chromatography on DEAE-Sepharose, gel filtration on AcA-44, and chromatography on blue-Sepharose. Rat uterine procollagenase appears as a protein doublet of Mr approximately 58,000, as indicated by two polyacrylamide gel electrophoresis systems, by AcA-44 chromatography, and by equilibrium sedimentation ultracentrifugal analysis. The proenzyme forms are converted by trypsin to an active enzyme doublet of Mr approximately 48,000. Small amounts of active enzyme, which are often generated during the purification, are electrophoretically indistinguishable from trypsin-activated collagenase. Active collagenase can be separated from the zymogen forms by DEAE-Sepharose chromatography. The two forms of the proenzyme doublet can be partially separated by gel filtration on AcA-44 and preliminary analysis indicates each has equal collagenolytic activity. The amino acid analysis of rat uterine collagenase reveals it to be markedly different from two other vertebrate collagenases whose composition is known. The uterine proenzyme is unusually rich in glycine and in the hydroxy amino acids and is considerably more acidic than the human skin fibroblast collagenase, consistent with the different ion-exchange behavior of the two molecules. The specific activity of rat uterine collagenase at 37 degrees C is approximately 3000 micrograms collagen/min/mg, using native reconstituted guinea pig skin type I collagen fibrils as substrate. The enzyme cleaves denatured collagen, but fails to attack a variety of noncollagen proteins.

Modification of the Stat1 SH2 Domain Broadly Improves Interferon Efficacy in Proportion to p300/CREB-binding Protein Coactivator Recruitment

A normal level of interferon (IFN) responsiveness via the Stat1 transcription factor is critical to the host, since decreased Stat1 signaling causes immune compromise and increased signaling is associated with inflammatory and neoplastic disease. Here we report how this balance may be influenced by novel alterations in the efficiency of Stat1 signaling. To enable disulfide-dependent and spontaneous formation of active Stat1 homodimer (as was done previously for Stat3), we engineered Stat1-CC with double-cysteine substitutions in the Src homology 2 (SH2)-homodimerization domain (at Ala-656 and Asn-658). In this case, however, mutant and wild-type Stat1 exhibited no difference inspontaneousdimerization. Moreover, Stat1-CC still required ligand-dependent Tyr-701 phosphorylation for function and exhibited hyperresponsiveness to IFN-β (that depends on Stat1/Stat2 heterodimerization) as well as IFN-γ (that depends on Stat1/Stat1 homodimerization). Hyperresponsivenss of Stat1-CC was accompanied by increased capacities for Tyr-701 phosphorylation and DNA binding, but these features were also found in a similarly substituted serine mutant (Stat1-SS) that showed no hyperresponsiveness to IFN-γ. This finding raised the possibility that SH2 domain mutations also influence downstream transcriptional efficiency. Indeed, each of these mutations also enhanced recruitment of the normally rate-limiting p300/CREB-binding Protein (CBP) coactivator to the transcriptional complex in proportion to the level of IFN-driven transactivation and gene expression. Additional modifications indicated that the mutant residues in the SH2 domain appeared to cooperate with Ser-727 in the C-terminal domain to regulate p300/CBP interaction with Stat1. The profile of IFN responsiveness translated into the same progressive increase in the level of viral clearance from Stat1- to Stat1-SS- to Stat1-CC-expressing cells. Thus, SH2 domain determinants may be modified to direct better Stat1 phosphorylation, DNA binding, and coactivator recruitment to fully improve IFN efficacy.

Self-cleavage of Human CLCA1 Protein by a Novel Internal Metalloprotease Domain Controls Calcium-activated Chloride Channel Activation

Background: CLCA proteins activate CaCCs; CLCAs have roles in cancer and inflammatory lung diseases, but their mechanism of action is unknown. Results: CLCA proteins must undergo self-cleavage via their own novel metalloprotease domain in the N terminus to activate CaCCs. Conclusion: Self-cleavage unmasks the N-terminal fragment, which alone activates CaCCs. Significance: This work identifies a unique ion channel activation mechanism defining framework to understand CLCA functions in diseases. The chloride channel calcium-activated (CLCA) family are secreted proteins that regulate both chloride transport and mucin expression, thus controlling the production of mucus in respiratory and other systems. Accordingly, human CLCA1 is a critical mediator of hypersecretory lung diseases, such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis, that manifest mucus obstruction. Despite relevance to homeostasis and disease, the mechanism of CLCA1 function remains largely undefined. We address this void by showing that CLCA proteins contain a consensus proteolytic cleavage site recognized by a novel zincin metalloprotease domain located within the N terminus of CLCA itself. CLCA1 mutations that inhibit self-cleavage prevent activation of calcium-activated chloride channel (CaCC)-mediated chloride transport. CaCC activation requires cleavage to unmask the N-terminal fragment of CLCA1, which can independently gate CaCCs. Gating of CaCCs mediated by CLCA1 does not appear to involve proteolytic cleavage of the channel because a mutant N-terminal fragment deficient in proteolytic activity is able to induce currents comparable with that of the native fragment. These data provide both a mechanistic basis for CLCA1 self-cleavage and a novel mechanism for regulation of chloride channel activity specific to the mucosal interface.

Prostaglandin H Synthase and Lipoxygenase Gene Families in the Epithelial Cell Barriera

Epithelial barrier cells (in skin, gut, and airway) are both active modulators and important targets of the inflammatory response, and some of these cellular events may be regulated at a molecular level by products of phospholipid-arachidonic acid metabolism. Accordingly, we have defined some of the characteristics of gene expression and enzyme regulation for distinct members of the PGH synthase and lipoxygenase gene families in normal and inflamed epithelial tissues and in epithelial cells isolated from mucosal and epidermal tissue (Table 1). A unifying scheme for our findings includes the following enzymatic systems: (i) a PGH synthase-1/PG isomerase pathway responsible for constitutive generation of prostaglandins (e.g., PGE2) and maintenance of physiologic epithelial function; (ii) a PGH synthase-2/PG isomerase and synthase pathway capable of producing additional prostaglandins (e.g., excess PGE2 and/or PGF2 alpha and PGD2) especially after stimulation by growth factors and cytokines; and (iii) a family of arachidonate 12- and 15-lipoxygenases that may serve to generate hydroxy acids (e.g., 12- and 15-HETE) as mediators of basal epithelial function and that (after overexpression and oxidant activation) may also catalyze membrane peroxidation that contributes to epithelial damage during inflammation. The regulatory mechanisms inherent in the control of this scheme provide a biochemical rationale for balancing constitutive and inducible oxygenation activities and maintaining epithelial barrier function.