Kyonggeun Yoon

Tissue specificity and developmental expression of rat osteopontin

Osteopontin is a 44 kd phosphoprotein abundant in bone matrix. We isolated a partial length cDNA for rat osteopontin and used it to examine its tissue specificity, its expression during bone development and its hormonal regulation. Osteopontin mRNA is most abundant in bone but is also found in considerable amounts in kidney. Osteopontin mRNA is regulated by the osteotropic hormones dexamethasone and 1,25(OH)2D3. Estimates of osteopontin mRNA levels indicate that the osteopontin gene is turned on relatively late in calvarial development.

Effects of Acidic and Basic Fibroblast Growth Factors on Osteoblastic Cells

Acidic (a) and basic (b) fibroblast growth factors (FGFs) are two related mitogenic and angiogenic factors. They are multifunctional in that they can affect proliferation and induce or delay differentiation. Both aFGF and bFGF were shown to stimulate proliferation of calvaria cells in situ as well as osteoblast-enriched calvaria-derived cells. bFGF was also found to suppress the expression of alkaline phosphatase, parathyroid hormone stimulatable adenylate cyclase, osteocalcin, and type I collagen in the osteoblastic ROS 17/2.8 cells. To explore a possible role for guanine nucleotide binding proteins we assessed the effects of pertussis toxin (PT) on FGF action. PT had opposite effects to those of bFGF on all parameters examined.

Alkaline phosphatase cDNA transfected cells promote calcium and phosphate deposition.

The full length cDNA of rat alkaline phosphatase (AP) was placed under the control of the SV40 early promoter. This plasmid was transfected by the calcium phosphate method into AP negative ROS 25/1 cells. Ten clones with AP specific activities ranging between 0.1-2 mumole/min/mg were isolated by cotransfection with the plasmid pSV2Neo, which renders the cells resistant to the antibiotic G418. Two clones with different AP specific activities: C (0.01 mumole/min/mg) and S (2.0 mumole/min/mg) and the osteoblastic ROS 17/2.8 cells (2.0 mumole/min/mg), were examined for their ability to mineralize. In vitro mineralization was tested by culturing cells in alpha-MEM containing 10% fetal bovine serum and 50 micrograms/ml ascorbate in the presence or absence of 10 mM beta-glycerophosphate. Mineralized deposits were observed in all cultures of the S clone and ROS 17/2.8 cells in the presence of beta-glycerophosphate, but not in C clones. Measurement of calcium and phosphorus levels in cells correlated with AP levels of transfected cells. However, extent of mineral accumulation in the transfected ROS 25/1 cells differ from the osteogenic ROS 17/2.8 cells. This finding indicates that high levels of AP may be a necessary constituent for the mineralization process together with other factors yet to be identified.

Alkaline phosphatase as a reporter enzyme.

This study examines the use of alkaline phosphatase (AP) as a reporter enzyme. We constructed a plasmid containing the cDNA which encodes the bone/liver/kidney rat AP under the control of the simian virus 40 (SV40) early promoter and used it to transfect Chinese hamster ovary, SV40-transformed African Green Monkey kidney 7, and rat osteosarcoma 25/1 mammalian cells. AP activity in these cells, measured three days later, was 40-400-fold above background. When AP and chloramphenicol acetyltransferase (CAT) plasmids were cotransfected, the detection of AP activity by a simple spectrophotometric assay was at least as sensitive as the detection of CAT activity using a radioactive substrate. Moreover, since mammalian AP is a membrane-bound ectoenzyme, transfected cells can be visualized by histochemical staining. This approach was used to estimate transfection efficiency. The convenient methods for AP detection should make it a useful reporter enzyme.

SV-40 Large-T Immortalization of Embryonic Bone Cells: Establishment of Osteoblastic Clonal Cell Lines

Cells derived from embryonic rat calvariae were immortalized by retroviral delivery of cDNA for the SV-40 large T antigen and the bacterial neomycin resistance gene. After selection with G418, cells were cloned by limiting dilution and screened for expression of osteoblast characteristics. One clone (RCT-3), derived from cells collected during the third period of enzymatic digestion, showed high constitutive expression of alkaline phosphatase (ALP), synthesized type I collagen in the virtual absence of type III and exhibited a parathyroid hormone (PTH)-responsive adenylate cyclase (EC50, 10 nM). Messenger RNAs for osteonectin and osteopontin were present in RCT-3 cells and osteopontin mRNA was enhanced by 1,25 (OH)2 vitamin D3 treatment. The other cell line (RCT-1), derived from cells released during the first 10 min of digestion, expressed osteoblast features only after 3 d treatment with 1 microM retinoic acid (RA). ALP activity increased from 0.003 to 0.25 mumole/min/mg protein, there was a substantial increase in the steady-state level of type I collagen mRNA and a dose-dependent and saturable response to PTH was induced (EC50, 10 nM). Osteopontin mRNA was induced by 1,25 (OH)2D3. This study has provided two new cell lines which may be useful models for studies of differentiation-related gene expression in bone cells.