C1Q-INDUCED CA2+RESPONSES IN FIBROBLAST HETEROGENEITY

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Human gingival fibroblast (HGF) function differently in health and disease. Our goal is to understand the cellular and molecular mechanisms that control fibroblast proliferation in wound healing. For this purpose we have established an in vitro model consisting of isolated, stable, phenotypically and metabolically characterized HGF subtypes of normal and granulation tissues with unique receptors for inflammatory complement C1q. C1q consists of two important regions: a collagen-like domain and a globular domain. HGF may be divided into subtype cC1qR of healthy gingiva expressing mainly receptors for C1q collagen-like domain, and subtype gC1qR of granulation tissue expressing mainly receptors for C1q globular domain. In vivo C1q selectively accumulates at sites of injury. In vitro C1q inhibits 60 to 80 % proliferation of HGF, suggesting an important role for this protein as a regulator of growth factors activities. The growth response of gC1qR cells is inhibited by C1q only during the G1 phase of the cell cycle; in contrast C1q inhibition of growth of cC1qR cells is cell cycle-independent. Subtypes differ in the pattern of cytosolic Ca2+ concentration following exposure to C1q. We hypothesize that the differences in intracellular Ca2+ response induced by the binding of globular versus collagen-like domain of C1q to specific fibroblast receptors underlie the different growth-factor response of subtypes, and that this mechanism may be important in the shifts of cellular populations and changes of metabolic activities of oral tissues during healing. The following aims verify how intracellular signals activated by C1q may regulate the growth-factor response of cells of normal and granulation tissues. For each subtype activated by C1q, Aim I quantifies levels of second messengers cAMP and inositol-phosphate turnover that mobilize and regulate Ca2+ flux from both external and internal sources, and assesses activation of protein kinases A and C. Aim II determines the role of these second messengers in the inhibitory effects of C1q on cell growth, and the regulation of PDGF receptor expression and function by C1q. Aim Ill characterizes the kinetics of C1q-induced Ca2+ influx/efflux in both resting and growth factor activated subtypes, determines activation of specific Ca2+channels on membrane of subtypes and activation of specific intracellular Ca2+stores. Results will elucidate significance of interactions of fibroblast subtypes with complement c1q at injured sites, relationship of subtypes to one another in wound healing, and may in part explain the shifts in cellular populations of healthy and diseased periodontium. These studies may lead to a new approach or adjunct to therapies that use growth factors to enhance tissue regeneration, by designing methodology that up- or down-regulates in the subtype of choice those elements of growth- factor signaling cascade that are affected by the activities of C1q present at sites of treatment.