Kinetics and interplay of mediators of inflammation-induced bone damage in the course of adjuvant arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation, bone erosion, and cartilage destruction in the joints. It is increasingly being realized that inflammation might play an important role in inducing bone damage in arthritis. However, there is limited validation of this concept in vivo in well-controlled experimental conditions. We addressed this issue using the adjuvant arthritis (AA) model of RA. In AA, the draining lymph nodes are the main sites of activation of pathogenic leukocytes, which then migrate into the joints leading to the induction of arthritis. We tested the temporal kinetics of mediators of bone damage [e.g., receptor activator of nuclear factor kappa-B ligand (RANKL), osteoprotegerin (OPG) and osteopontin (OPN)] and inflammation (pro-inflammatory cytokines and chemokines) in the draining lymph node cells (LNC) at different phases of AA, and then examined their inter-relationships. Our study revealed that, together with cytokines/chemokines, some of the mediators of bone remodeling are also produced in LNC. Various cytokines/chemokines showed distinct kinetics of expression as well as patterns of correlation with mediators of bone remodeling at different phases of the disease. Pro-inflammatory cytokines such as TNF-alpha are known to play an important role in bone damage. Interestingly, there was a positive correlation between TNF-alpha and RANKL, between RANKL and each of the 3 chemokines tested (RANTES, MIP-1alpha, and GRO/KC), and between TNF-alpha and RANTES. Our results in the AA model lend support to the concept of osteo-immune crosstalk during the course of autoimmune arthritis.

Inhibition of Na+/Ca2+ exchange with KB-R7943 or bepridil diminished mineral deposition by osteoblasts.

Osteoblasts form new bone by secreting a complex extracellular matrix that has the capacity to mineralize when adequate amounts of calcium and phosphate are supplied. The studies reported here show that long-term treatment of cultured, primary osteoblasts with Na+/Ca2+ exchanger (NCX) inhibitors, bepridil and KB-R7943, impacts in a dose-dependent manner the ability of the cells to form a calcified matrix. Treatment of confluent osteoblast cultures for 14 days with low levels of bepridil (3.0 microM) or KB-R7943 (1.0 microM and 0.1 microM) resulted in a significantly diminished capacity of these cells to mineralize bone matrix, without significantly altering cell morphology, viability, or cell differentiation. The data indicate that inhibition of NCX reduces mineral accumulation in the bone matrix by blocking the efflux of Ca2+ from the osteoblast into the bone fluid. In addition, immunocytochemistry of type I collagen (COLI) and bone sialoprotein (BSP) suggests that inhibition of NCX by 1.0 microM KB-R7943 also may impair the secretion of bone matrix proteins by the osteoblasts. This study is the first to show that NCX is an important regulator of the bone fluid microenvironment and that NCX appears critical to the mineralization process.

Asymmetric distribution of functional sodium-calcium exchanger in primary osteoblasts.

To understand calcium translocation in osteoblasts, we have determined the location of sodium-calcium (Na-Ca) exchanger (NCX) in relation to actin and alpha-tubulin in primary cultures of avian osteoblasts. Osteoblasts derived from the periosteal surface of tibias from growing chickens were cultured for 8 days in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Lysates immunoblotted with antibodies raised against the canine cardiac Na-Ca antibodies revealed a 70 kDa exchanger protein. Cross-reactivity of the anti-NCX antibody was confirmed by enriching for NCX in protein samples derived from plasma membrane vesicles by affinity chromatography using the exchanger inhibitory peptide. Fractions enriched for the exchanger were eluted from the column and subjected to immunoblotting with the anti-NCX antibody, revealing an intense single band at 70 kDa. Examination of live cells loaded with Calcium Green-1 AM ester by confocal microscopy demonstrated sodium-dependent calcium uptake, confirming the presence of functional NCX in intact cells. Immunolocalization studies of osteoblasts stained with anti-NCX antibodies revealed asymmetric localization of the exchanger in cultured osteoblasts, residing almost entirely within two 0.5-microm optical sections along the substrate adherent side of the cells. Since NCX is known to be a low-affinity, high-capacity calcium translocating molecule and also appears to be asymmetrically positioned, it is likely to play a key role in bone formation.

Characterization of a pollen-expressed gene encoding a putative pectin esterase of Petunia inflata.

From a pollen tube cDNA library of Petunia inflata, we isolated cDNA clones encoding a protein, PPE1, which exhibits sequence similarity with plant, bacterial, and fungal pectin esterases. Genomic clones containing the PPE1 gene were isolated using cDNA for PPE1 as a probe, and comparison of the cDNA and genomic sequences revealed the presence of a single intron in the PPE1 gene. During pollen development, PPE1 mRNA was first detected in anthers containing uninucleate microspores; it reached the highest level in mature pollen and persisted at a high level in in vitro germinated pollen tubes. The observed expression pattern of the PPE1 gene suggests that its product may play a role in pollen germination and/or tube growth.