Fibroblast proliferation and procollagen production are central features of tissue repair and fibrosis. In addition to its role in blood clotting, the coagulation cascade proteinase thrombin can contribute to tissue repair by stimulating fibroblasts via proteolytic activation of proteinase-activated receptor-1 (PAR1). During hemostasis, the coagulation cascade proteinase factor X is converted into factor Xa. We have previously shown that factor Xa upregulates fibroblast proliferation via production of autocrine PDGF. In this study, we further examined the effects of factor Xa on fibroblast function and aimed to identify its signaling receptor. We showed that factor Xa stimulates procollagen promoter activity and protein production by human and mouse fibroblasts. This effect was independent of PDGF and thrombin production, but dependent on factor Xa proteolytic activity. We also showed that PAR1-deficient mouse fibroblasts did not upregulate procollagen production, mobilize cytosolic calcium, or proliferate in response to factor Xa. Desensitization techniques and PAR1-specific agonists and inhibitors were used to demonstrate that PAR1 mediates factor Xa signaling in human fibroblasts. This is the first report that factor Xa stimulates extracellular matrix production. In contrast with endothelial cells and vascular smooth muscle cells, fibroblasts appear to be the only cell type in which the effects of factor Xa are mediated mainly via PAR1 and not PAR2. These findings are critical for our understanding of tissue repair and fibrotic mechanisms, and for the design of novel approaches to inhibit the profibrotic effects of the coagulation cascade without compromising blood hemostasis.

Abstract. In the rat optic nerve, bipotential O-2A progenitor cells give rise to oligodendrocytes and type 2 astrocytes on a precise schedule. Previous studies suggest that PDGF plays an important part in timing oligodendrocyte development by stimulating O-2A progenitor cells to proliferate until they become mitotically unresponsive to PDGE stop dividing, and differentiate automatically into oligodendrocytes. Since the loss of mitotic responsiveness to PDGF has been shown not to be due to a loss of PDGF receptors, we have now examined the possibility that the unresponsiveness results from an uncoupling of these receptors from early intracellular signaling pathways.We show that (a) although PDGF does not stimulate newly formed oligodendrocytes to synthesize DNA, it induces an increase in cytosolic Ca 2+ in these cells; (b) a combination of a Ca 2+ ionophore plus a phorbol ester mimics the effect of PDGF, both in stimulating O-2A progenitor cell division and in reconstituting the normal timing of oligodendrocyte differentiation in culture; and (c) the same combination of drugs does not stimulate newly formed oligodendrocytes to proliferate, even in the presence of PDGF or dibutyryl cAMP. The most parsimonious explanation for these results is that O-2A progenitor cells become mitotically unresponsive to PDGF because the intracellular signaling pathways from the PDGF receptor to the nucleus are blocked downstream from the receptor and some of the early events that are triggered by receptor activation.

The role(s) of the novel stargazin-like ␥-subunit proteins remain controversial. We have shown previously that the neuron-specific ␥7 suppresses the expression of certain calcium channels, particularly Ca V 2.2, and is therefore unlikely to operate as a calcium channel subunit. We now show that the effect of ␥7 on Ca V 2.2 expression is via an increase in the degradation rate of Ca V 2.2 mRNA and hence a reduction of Ca V 2.2 protein level. Furthermore, exogenous expression of ␥7 in PC12 cells also decreased the endogenous Ca V 2.2 mRNA level. Conversely, knockdown of endogenous ␥7 with short-hairpin RNAs produced a reciprocal enhancement of Ca V 2.2 mRNA stability and an increase in endogenous calcium currents in PC12 cells. Moreover, both endogenous and expressed ␥7 are present on intracellular membranes, rather than the plasma membrane. The cytoplasmic C terminus of ␥7 is essential for all its effects, and we show that ␥7 binds directly via its C terminus to a heterogeneous nuclear ribonucleoprotein (hnRNP A2), which also binds to a motif in Ca V 2.2 mRNA, and is associated with native Ca V 2.2 mRNA in PC12 cells. The expression of hnRNP A2 enhances Ca V 2.2 I Ba , and this enhancement is prevented by a concentration of ␥7 that alone has no effect on I Ba . The effect of ␥7 is selective for certain mRNAs because it had no effect on ␣2␦-2 mRNA stability, but it decreased the mRNA stability for the potassium-chloride cotransporter, KCC1, which contains a similar hnRNP A2 binding motif to that in Ca V 2.2 mRNA. Our results indicate that ␥7 plays a role in stabilizing Ca V 2.2 mRNA.

1 The pharmacological characteristics of a putative Ca2" activated K+ channel (IKCa channel)

During terminal differentiation, keratinocytes lose the ability to divide. One indicator of responsiveness to certain growth factors is a transient rise in the intracellular concentration of free calcium ions ([Ca2+]i). The aim of our experiments was to discover whether or not terminally differentiating keratinocytes have lost the ability to exhibit an increase in [Ca2+]i in response to factors that stimulate [3H]thymidine incorporation and increase [Ca2+]i in undifferentiated keratinocytes. [Ca2+]i was measured with the calcium indicator dye FURA-2 and by a ratio imaging method. Expression of involucrin, a precursor of the keratinocyte cornified envelope, was used as a marker of terminal differentiation. Measurements were made on stratified colonies of cells grown in standard medium (containing 1.8 mM calcium ions) and on cell monolayers in low calcium medium (0.1 mM). Treatment of serum-starved monolayers with substance P, bombesin or complete growth medium containing 10% fetal calf serum resulted in increased [3H]thymidine incorporation. A switch from low calcium to standard medium also stimulated [3H]thymidine incorporation whether or not the cells had been serum-starved. In each experiment some cells showed an increase in [Ca2+]i while others did not. However, the heterogeneity in the [Ca2+]i response did not reflect the terminal differentiation status of individual cells: both involucrin-positive and -negative cells were found in the responding and nonresponding populations. Involucrin-positive and -negative areas of stratified cultures also underwent a transient increase in [Ca2+]i in response to serum-containing medium. Our data therefore indicate that both proliferating (involucrin-negative) and post-mitotic, terminally differentiating (involucrin-positive) keratinocytes can respond to mitogenic stimuli by an increase in [Ca2+]i.(ABSTRACT TRUNCATED AT 250 WORDS)