Abstract. Mechanical force regulates gene expression and cell proliferation in a variety of cell types, but the mechanotransducers and signaling mechanisms involved are highly speculative. We studied the fibroblast signaling mechanism that is activated when cells are switched from mechanically stressed to mechanically relaxed conditions, i.e., stress relaxation. Within I0 min after initiation of stress relaxation, we observed a transient 10-20-fold increase in cytoplasmic cyclic AMP (cAMP) and a threefold increase in protein kinase A activity. The increase in cAMP depended on stimulation of adenylyl cyclase rather than inhibition of phosphodiesterase. Generation of cAMP was inhibited by indomethacin, and release of arachidonic acid was found to be an upstream step of the pathway. Activation of signaling also depended on influx of extracellular Ca 2+ because addition of EGTA to the incubations at concentrations just sufficient to exceed Ca 2+ in the medium inhibited the stress relaxation-dependent increase in free arachidonic acid and cAMP. This inhibition was overcome by adding CaC12 to the medium. On the other hand, treating fibroblasts in mechanically stressed cultures with the calcium ionophore A23187-stimulated arachidonic acid and cAMP production even without stress relaxation. In summary, our results show that fibroblast stress relaxation results in activation of a Ca2+-dependent, adenylyl cyclase signaling pathway. Overall, the effect of stress relaxation on cAMP and PKA levels was equivalent to that observed after treatment of cells with forskolin.

Wound fluid contains several proteinases that are important in the repair process. In this study, we analyzed caseinolytic activity in wound fluid obtained from acute (burn) wounds. Caseinolytic activity in wound fluid increased markedly 2 d after injury and appeared on casein zymographs as a series of bands or a smear ranging from 30 to 100 kDa. Most of the enzyme activity was inhibited by the synthetic human neutrophil elastase inhibitor MDL 27,367 but not by the naturally occurring inhibitor of elastase, human secretory leukoproteinase inhibitor. Fractionation of wound fluid indicated that a single enzyme accounted for approximately 80% of the caseinolytic activity. This enzyme degraded the elastase substrate methoxysuccinyl-ala-ala-pro-val-p-nitroanilide at a slow rate. The above findings suggested that the enzyme responsible for caseinolytic activity might be proteinase 3, an elastase-related enzyme whose physiologic functions are poorly understood. Consistent with the above possibility, we found that monoclonal antibodies against proteinase 3 removed caseinolytic activity from wound fluid, and that purified proteinase 3 had a similar caseinolytic profile and inhibitor sensitivity to burn fluid.

Abstract. Fibroblast contraction of stressed collagen matrices results in activation of a cAMP signal transduction pathway. This pathway involves influx of extracellular Ca 2+ ions and increased production of arachidonic acid. We report that within 5 min after initiating contraction, a burst of phosphatidic acid release was detected. Phospholipase D was implicated in production of phosphatidic acid based on observation of a transphosphatidylation reaction in the presence of ethanol that resulted in formation of phosphatidylethanol at the expense of phosphatidic acid. Activation of phospholipase D required extracellular Ca 2+ ions and was regulated by protein kinase C. Ethanol treatment of cells also inhibited by 60-70% contraction-dependent release of arachidonic acid and cAMP but had no effect on increased cAMP synthesis after addition of exogenous arachidonic acid or on phospholipase A2 activity measured in cell extracts. Moreover, other treatments that inhibited the burst of phosphatidic acid release after contraction--chelating extracellular Ca 2+ or downregulating protein kinase C--also blocked contraction activated cyclic AMP signaling. These results were consistent with the idea that phosphatidic acid production occurred upstream of arachidonic acid in the contraction-activated cAMP signaling pathway.IVING organisms can sense and react to mechanical stimuli, although the underlying regulatory mechanisms are not yet well understood. A variety of studies have shown that many types of cells respond to mechanical stress by an increase in cell mass or number, and that mechanical signals are important determinants of cell differentiation (Ryan, 1989;Thyberg et al