Interleukin-1 inhibits the synthesis of collagen by fibroblasts

Human dermal fibroblasts, exposed to human or porcine Interleukin-1, responded by an inhibition of collagen synthesis in a dose dependent manner. Incubation with Il-1 for more than 8 h was required to see an appreciable effect. The phenomenon was not dependent on the presence of serum in the culture medium. Since a stimulation of prostaglandin E2 secretion was also observed in presence of Il-1, we investigated the eventual role of arachidonic acid metabolites in the phenomenon. Inhibitors interfering with arachidonate metabolism, namely indomethacin, acetyl salicylic acid, BW 755 C and NDGA had no influence on the inhibition of collagen synthesis caused by Il-1. These data suggest that both cyclooxygenase and lipoxygenase derived metabolites of arachidonic acid are unlikely to play a role in the mechanism.     

Kinetic characterization of the normal and detergent-perturbed reaction cycles of the sarcoplasmic reticulum calcium pump. Rate-limiting step(s) under different conditions

We previously characterized the structural features of the interaction of sarcoplasmic reticulum membranes with nonsolubilizing concentrations of C12E8, the non-ionic detergent octaethylene glycol monododecyl ether (Andersen, J.P., le Maire, M., Kragh-Hansen, V., Champeil, P., and M?ller, J. V. (1983) Eur. J. Biochem. 134, 205-214). The present study characterizes especially the functional aspects and implications of the detergent-induced perturbation for an understanding of ATPase function. Perturbing detergent decreased Vmax, but left Ca2+ transport intact. Detergent incorporation affected neither the calcium-dependent phosphorylation from ATP, as judged from multimixer quenching experiments, nor the calcium-releasing transition between the two phosphoenzyme forms (Ca2E1P to E2P), as judged from kinetically resolved dual-wavelength measurements with the calcium-sensitive dye antipyrylazo III. However, the decrease in Vmax was accounted for by a decrease in the rate of enzyme dephosphorylation by a factor of 3-4, whereas the Ca2+-dependent transition between the nonphosphorylated enzyme forms (E2 to Ca2E1) was enhanced almost 10-fold. Evidence of a conformational change of E2 by C12E8 toward that of the E1 state to account for the perturbed reactions was obtained from experiments on vanadate reactivity and tryptic degradation pattern. Both direct and steady-state evidence was obtained for an acceleration by ATP of the Ca2E1P to E2P transition which may account for the low affinity modulatory effect of the nucleotide on enzyme turnover. The kinetic data indicated that reduction of ATP hydrolysis by C12E8 coincided with conditions where E2P dephosphorylation becomes rate-limiting (high ATP concentration, low pH, absence of potassium). Otherwise, the Ca2E1P to E2P transition is deduced to be a rate-limiting step for the ATPase cycle, whereas the potential for rate control of the cycle by modulation of the E2 to Ca2E1 transition is very small. Only in special circumstances (absence of potassium, high temperature, and using ITP as a substrate) did this transition become a rate-limiting step, subject to rate enhancement of the whole cycle by detergent perturbation.     

Induction of interleukin-1 beta production in human dermal fibroblasts by interleukin-1 alpha and tumor necrosis factor-alpha. Involvement of protein kinase-dependent and adenylate cyclase-dependent regulatory pathways.

It has previously been demonstrated that interleukin-1 (IL-1) is expressed in a variety of fibroblast cell lines. In this study, we investigated the mechanisms involved in the regulation of IL-1 beta production by cultured human dermal fibroblasts. We have shown that IL-1 beta is constitutively expressed as a cell-associated form, with no soluble form detectable in control cell or in stimulated cell supernatants. IL-1 alpha and tumor necrosis factor-alpha (TNF-alpha) exerted a dose-dependent stimulation on the production of the cell-associated IL-1 beta, as estimated using a specific enzyme linked immunosorbent assay (ELISA). As expected, this effect was accompanied by a huge release of prostaglandin E2 (PGE2) and a transient rise in intracellular cyclic AMP. Furthermore, IL-1 beta production was elevated to a lesser extent by the addition of increasing concentrations of the protein kinase C activator phorbol myristate acetate or by low concentration (0.001 microgram/ml) of PGE2. In contrast, higher concentrations (0.1 and 1 micrograms/ml) of PGE2, as well as exogenous dibutyryl-cyclic AMP, were clearly inhibitory. H7, an inhibitor of protein kinases also reduced the stimulatory effect of IL-1 alpha and TNF-alpha. Together with the results obtained with phorbol myristate acetate, these data suggest that protein kinase C may play a role in the upregulation of IL-1 beta expression in normal skin fibroblasts. The addition of indomethacin not only suppressed prostaglandin synthesis, but also dramatically reduced cyclic AMP formation, probably because the PGE2-induced stimulation of adenylate cyclase was abolished. This resulted in a strong potentiation of the stimulatory effect of IL-1 alpha and TNF-alpha, supporting the role of both the cyclooxygenase and adenylate cyclase pathways in the endogenous downregulation of IL-1 beta induction by the two cytokines studied.     

pH and magnesium dependence of ATP binding to sarcoplasmic reticulum ATPase. Evidence that the catalytic ATP-binding site consists of two domains

Nucleotide binding to sarcoplasmic reticulum vesicles was investigated in the absence of calcium using both filtration and fluorescence measurements. Filtration assays of binding of radioactive nucleotides at concentrations up to 0.1 mM gave a stoichiometry of one ATP-binding site/sarcoplasmic reticulum ATPase molecule. When measured in the presence of calcium under otherwise similar conditions, ATPase velocity rose 4-8-fold (depending on pH and magnesium concentration) when the ATP concentration was increased from 1 microM to 0.1 mM. Binding of ATP and ADP enhanced the intrinsic fluorescence of sarcoplasmic reticulum ATPase, but AMP and adenosine did not affect it. Both filtration and fluorescence measurements showed that binding of metal-free ATP is independent of pH (Kd = 20-25 microM) but that the presence of magnesium induces pH dependence of the binding of the Mg.ATP complex (Kd = 10 microM at pH 6.0 and 1.5 microM at pH 8.0). Binding of metal-free ADP was pH-dependent but was not affected by magnesium. High magnesium concentrations inhibited nucleotide binding. These results suggest that ATP interacts with two different domains of Ca-ATPase that form the catalytic site. The first domain may bind the adenine moiety of the substrate, and the pH dependence of ADP binding suggests the participation of His683 in this region. The second domain of the catalytic site may bind the gamma-phosphate and the magnesium ion of the Mg.ATP complex and constitute the locus of the electrostatic interactions between the substrate and the enzyme.     

Primary ciliary dyskinesia.

Primary ciliary dyskinesia represents a group of heritable disorders of cilia and sperm affecting between 1 in 15,000 and 1 in 30,000 persons. Those affected lack measurable mucociliary clearance and suffer the constant misery of rhinorrhea and chronic productive cough. Because mucociliary clearance constitutes one of the respiratory system''s major lines of defense, these patients are vulnerable to chronic sinusitis, bronchitis, pneumonia, and otitis media. Left untreated, these problems may progress to bronchiectasis, found frequently in adult patients, or pulmonary hypertension with eventual cor pulmonale. Screening for this disorder includes some simple and inexpensive methods as well as more exotic techniques requiring special camera equipment and an electron microscope to make a definitive diagnosis. Physiotherapy techniques can be taught to patients with primary ciliary dyskinesia and go a long way toward making up for the lack of mucociliary clearance. Vigorous bronchopulmonary toilet and palliative measures may enable these patients to enjoy relatively normal lives.     

A direct fluorescence study of the transient steps induced by calcium binding to sarcoplasmic reticulum ATPase

The sarcoplasmic reticulum intrinsic fluorescence level was closely correlated with the ATPase functional state, from pH 5.5 to 8.5. The fluorescence signal was used in stopped flow measurements for direct study of transient pump kinetics after calcium binding or removal. The signal change time course, which depends solely on the free calcium concentration in the observation chamber, was analyzed as a single exponential. Rate constants (kobs) were relatively slow (5 to 20 s-1), indicating multistep interaction between calcium and the transport protein. At pH 7 and 20 degrees C, and in the presence of 100 mM potassium and 1 to 20 mM MgCl2, kobs first decreased, and then increased as the calcium concentration rose. Similar experiments were performed at pH 6. Data were analyzed according to a scheme in which sarcoplasmic reticulum . calcium complex formation is controlled by a slow isomerization step occurring either before or after the rapid calcium binding to the high affinity site. The results are discussed with reference to published rapid quenching experiments. Under our conditions, i.e. in the absence of a calcium gradient across the membrane, the calcium pump cycle step in which reorientation of the calcium binding sites occurs cannot be identified with the isomerization step mentioned above.     

How to model the events in cutaneous fibrosis?

Skin fibrosis is classically seen as the consequence of chronic inflammation and altered healing response that is characterized by the differentiation of fibroblasts into secretory myofibroblasts and accumulation of connective tissue. Although fibrosis severely affects organ function and causes esthetic defects, no effective therapy is currently available to attenuate the fibrogenic process probably because the fibrogenic process is more complex than previously thought. Indeed, it might involve several interacting and mutually dependent cell types (fibroblasts, keratinocytes, endothelial cells, inflammatory cells), numerous paracrine factors, bio-active molecules and micro-environmental stimuli (growth factors, vasoactive peptides, balance between pro- and anti-inflammatory cytokines, coagulation system, reactive oxygen species, extracellular matrix...). In this perspective, the traditional approach that model individual cell response in simple cell culture system is probably inadequate and too simplistic. This article reviews the new models used to study skin fibrosis in vitro, in organotypic culture systems and in vivo and examines how these different models might be used to identify new molecular pathways involved in fibrogenesis. The monolayer cultures allow the study of fibrogenic signals induced by a single factor on a single cell type. Isolation of cells from fibrotic tissue allows to define the fibrogenic differentiation acquired in vivo. The organotypic models allow cell to cell and cell to matrix interaction and the experimental models in pigs and mice allowed studies in integrated physiological systems. These various and complementary models would also provide new tools to develop and test new drugs and treatments.     

Disruption of basal JNK activity differentially affects key fibroblast functions important for wound healing

We used both a gene knockout approach and pharmacologic modulation to study the implication of the JNK pathway in regulating fibroblast motility, capacity to contract mechanically unloaded collagen gels, and type I collagen gene expression in vitro. These parameters, which are important for tissue repair, are positively regulated by transforming growth factor (TGF)-beta, a cytokine viewed as playing a master role during wound healing. We demonstrate that basal JNK activity is critical for fibroblast motility because (a) mouse embryo jnk-/- fibroblasts exhibit significantly lower ability to close mechanically induced cell layer wounds than their wild-type (wt) counterparts, and (b) wound closure by human dermal fibroblasts is dramatically impaired by the specific JNK inhibitor SP600125. junAA fibroblasts, in which amino acids Ser63 and Ser73 of c-Jun are replaced by two Ala residues so that c-Jun cannot be phosphorylated by JNK, also exhibited impaired motility, suggesting that c-Jun phosphorylation by JNK is critical for fibroblast migration. In sharp contrast to their lesser motility on plastic, jnk-/- and junAA fibroblasts contracted free-floating, mechanically unloaded, collagen lattices markedly faster than wt fibroblasts. Furthermore, basal mRNA steady-state levels for types I and III collagen genes were similar in jnk-/- and wt fibroblasts. Likewise, overexpression of a dominant-negative mutant form of MKK4 in dermal fibroblasts did not affect collagen expression. We also demonstrate that basal JNK activity does not affect either TGF-beta-induced collagen gene expression or lattice contraction, whereas on the other hand, the blockage of motility initiated by JNK inhibition cannot be overcome by TGF-beta. Together these results demonstrate discrete, yet significant and highly specific, regulation of fibroblast functions important for wound healing by basal JNK activity.