Fibroblast heterogeneity of signal transduction mechanisms to complement-C1q. Analyses of calcium mobilization, inositol phosphate accumulation, and protein kinases-C redistribution.
Fibroblasts of healthy and granulation gingiva are phenotypically heterogeneous with regard to binding C1q collagen-like (cC1qR) or C1q globular-heads (gC1qR) regions, respectively. Here, isolated fibroblast subsets, expressing either the cC1qR or the gC1qR phenotype, were stimulated with C1q, and assessed for changes in cytosolic free calcium [Ca2+]i, accumulation of inositol trisphosphate (IP3), and redistribution of Ca2+-dependent protein kinases-C (cPKCs) from cytosol to membranes. Changes in [Ca2+]i were determined using Indo-1 fluorescence in combination with adhering cell analysis and sorting (ACAS) cytometry. Accumulation of IP3 was quantified using a competitive radioreceptor binding assay. Redistribution of cPKCs was evaluated by immunoblotting with antibodies to PKCalpha/betaI-betaII/gamma. Subsets manifested different fluctuations in [Ca2+]i levels 20 seconds after C1q-stimulation in the presence of millimolar concentrations of external calcium. Whereas cC1qR fibroblasts responded with a 38% over baseline [Ca2+]i increase which was sustained for 20 to 30 minutes, gC1qR fibroblasts responded with a higher (264% over baseline) and more rapid (2 to 3 minutes) transient. Likewise, subsets exhibited different kinetics of IP3 accumulation. Whereas cC1qR fibroblasts responded with an IP3 increase of 32 +/- 3 pmol/10(4) cells over baseline after 5 seconds stimulation, gC1qR fibroblasts responded after 15 to 20 seconds with a lower increase (13 +/- 0.8 IP3 pmol/10(4) cells over baseline). Subsets differed in cPKCs redistribution which peaked in gC1qR-membranes 30 seconds after stimulation and remained sustained between 10 and 30 minutes. No cPKC redistribution was detectable in stimulated cC1qR-cells. We conclude that fibroblasts are heterogeneous in phosphoinositide-Ca2+ signaling and cPKC redistribution to C1q, and suggest that these differences may affect activities of normal and granulation gingiva.
Bordin
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Department of Periodontics, University of Washington, Seattle 98195, USA. bordin@u.washington.edu
Costa
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Tan
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D000704:Analysis of Variance; D000906:Antibodies; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003094:Collagen / Q000378:metabolism; D015922:Complement C1q / Q000235:genetics / Q000378:metabolism*; D003165:Complement System Proteins / Q000235:genetics / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D005347:Fibroblasts / Q000201:enzymology / Q000378:metabolism*; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D015870:Gene Expression; D005881:Gingiva / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D006097:Granulation Tissue / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D006801:Humans; D007211:Indoles; D007295:Inositol Phosphates / Q000378:metabolism*; D010641:Phenotype; D011493:Protein Kinase C / Q000378:metabolism*; D015398:Signal Transduction; D013997:Time Factors
D000906:Antibodies; D005456:Fluorescent Dyes; D007211:Indoles; D007295:Inositol Phosphates; D015922:Complement C1q; D003094:Collagen; D003165:Complement System Proteins; D011493:Protein Kinase C; C048960:indo-1; D002118:Calcium