Calcium Sensing in Cultured Chondrogenic RCJ3.1C5.18 Cells*

Abstract

The availability of Ca2+ in the extracellular fluid plays an important role in regulating cartilage and bone formation. We hypothesized that chondrocytes detect changes in the extracellular [Ca2+] ([Ca2+]o) and modify their function. The effects of changing [Ca2+]o on the expression of matrix proteins were quantified by staining of cartilage nodules with alcian green and assessing RNA levels of cartilage-specific genes in chondrogenic RCJ3.1C5.18 (C5.18) cells. Alcian green staining in these cells decreased with increasing [Ca2+]o in a dose-dependent and reversible manner (ID50, approximately 2 mM Ca2+). RNA levels for aggrecan and type II collagen decreased with increasing [Ca2+]o (ID50, approximately 2.0 and 4.1 mM Ca2+, respectively). RNA levels for type X collagen and alkaline phosphatase were also reduced by high [Ca2+]o with ID50 values of approximately 2.9 and 1.6 mM Ca2+, respectively. These responses were rapid, in that increasing [Ca2+]o from 1.0 to more than 6 mM suppressed aggrecan RNA levels by about 50%, and lowering [Ca2+]o from 2.9 to 1.0 mM increased aggrecan RNA levels by about 300% within 4 h. As Ca2+ receptors (CaRs) mediate extracellular Ca2+ sensing in parathyroid and kidney, we assessed the expression of CaRs in these cells. C5.18 cells stained positively for CaR protein with an anti-CaR antiserum and for CaR RNA by in situ hybridization. An approximately 150-kDa protein was detected by immunoblotting with anti-CaR antiserum. CaR antisense oligonucleotides suppressed the expression of CaR protein and enhanced RNA levels of aggrecan in C5.18 cells. These data support the idea that CaRs are expressed in this cell system and may be involved in regulating chondrogenic gene expression.

Amino Acids in the Second and Third Intracellular Loops of the Parathyroid Ca2+-sensing Receptor Mediate Efficient Coupling to Phospholipase C

“…Fluorescein-conjugated goat-anti rabbit IgG for immunocytochemistry was obtained from Molecular Probes, Inc. (Eugene, OR). Other supplies were from previously noted sources (3,19).…”

“…Immunoblotting and Immunocytochemistry-Crude membrane protein fractions were prepared from HEK293 cells, electrophoresed on SDS-polyacrylamide electrophoresis gels, and transferred to nitrocellulose membranes as described (3,19). Membranes were blotted with one of two affinity-purified rabbit antisera (21825B, 50 nM or 321113A, 10 nM) (3,19), and signals were detected with an enhanced chemiluminescence (ECL) assay kit (Amersham Pharmacia Biotech).…”

“…Membranes were blotted with one of two affinity-purified rabbit antisera (21825B, 50 nM or 321113A, 10 nM) (3,19), and signals were detected with an enhanced chemiluminescence (ECL) assay kit (Amersham Pharmacia Biotech). Protein expression of all mutant CaR constructs was tested by immunoblotting at least twice along with wt controls.…”

“…Protein expression of all mutant CaR constructs was tested by immunoblotting at least twice along with wt controls. Immunocytochemistry with 3,3Ј-diaminobenzidene-and fluorescein-conjugated antibodies was performed on cells grown on chamber slides, fixed for 30 min, incubated with primary and secondary antibodies, and then stained as previously detailed (3,19). Primary antibody was either a CaR antiserum (21825A; 500 nM), this antiserum preincubated with 100-fold excess peptide, or non-immune rabbit serum.…”

“…Primary antibody was either a CaR antiserum (21825A; 500 nM), this antiserum preincubated with 100-fold excess peptide, or non-immune rabbit serum. The antiserum was raised against a peptide derived from the extracellular domain of the bovine parathyroid CaR (3,19). For 3,3Ј-diaminobenzidine-staining, slides were counterstained with aqueous hematoxylin.…”