Retinoic acid and tumour necrosis factor-alpha act in concert to control the level of alkaline phosphatase mRNA.

Retinoic acid has a specific role in cellular differentiation and is believed to act by regulating the transcription of specific genes. In the present work, evidence is provided to show that alkaline phosphatase (ALP) gene expression is mediated by retinoic acid in a model clonal cell line (UMR 201) derived from rat neonatal calvaria. These cells have the characteristics of relatively undifferentiated mesenchymal cells with a very low basal ALP activity which is dramatically increased by retinoic acid. Messenger RNA for ALP was clearly demonstrated when the cells were treated with 1 microM retinoic acid for 24 h. Recombinant human tumour necrosis factor-alpha (recombinant TNF-alpha) interacted with retinoic acid to potentiate the rise in ALP activity, although recombinant TNF-alpha alone had no effect. The potentiation of retinoic acid-induced ALP activity was correlated with an increased amount of mRNA for ALP with the combined treatment. By observing the rate of decay of mRNA for actin and ALP, we were able to demonstrate that the interaction between retinoic acid and recombinant TNF-alpha modulated the steady state of ALP mRNA. The mode of action of recombinant TNF-alpha may serve as a model for other paracrine regulators of cell function.

Regulation of alkaline phosphatase expression in a neonatal rat clonal calvarial cell strain by retinoic acid.

A clonal cell strain, UMR 201, was established from a culture of rat calvarial cells by the process of limiting dilution on a collagen substratum. One-day-old neonatal rat calvaria stripped of periosteum were placed on collagen in alpha-MEM with 10% fetal bovine serum (FBS). Cells that grew out from the calvaria were passaged eight times to select cells with the ability to proliferate in culture before cloning was attempted. Cells from the clonal strain were homogeneous in appearance with a doubling time in culture of about 24 hours. The UMR 201 cells formed predominantly type 1 collagen. When treated with retinoic acid (RA), all cells showed an intense staining for alkaline phosphatase (ALP). This effect of RA on the expression of ALP activity was reversible and was time and dose dependent. The earliest change was observed within 6 hours. In contrast, single and isolated clumps of untreated cells stained positively for ALP only when they were confluent. Coincubation with dactinomycin up to 3 hours after the addition of RA completely prevented the expression of ALP, whereas dactinomycin became progressively less effective when added at later times. This is interpreted as indicating a regulatory role of RA on the gene expression of ALP. Other hormones acting on bone, such as 1,25(OH)2 vitamin D3 and dexamethasone, also modulate ALP activity. The cells showed morphologic evidence of senescence after passage 12. Our preliminary studies showed that the UMR 201 cells had the characteristics of relatively undifferentiated mesenchymal cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin release from a cloned precursor beta cell line.

This paper describes the establishment in long-term tissue culture of a functional, clonal beta (B) cell line UMR 407/3 derived from neonatal rat pancreas. Immunofluorescence demonstrated specific and uniform staining for insulin. Transmission electron microscopy showed the presence of microvilli and cytoplasmic granules. The doubling time in culture was approximately 60 h in 2% (v/v) fetal calf serum with inhibition of growth at confluence. Biochemical studies demonstrated the incorporation of [3H]leucine into proinsulin and insulin, with insulin comprising 43.6% of the total radioactivity incorporated into immune complexes. When incubated at 37 degrees C for 30 min with Krebs-Ringer bicarbonate buffer (pH 7.4), the amount of insulin released on stimulation by 16.7 mmol glucose/l, 20 mmol DL-glyceraldehyde/l or 20 mmol alpha-ketoisocaproate/l was significantly higher compared with 5.6 mmol glucose/l. The mean insulin content was equivalent to 99 +/- 0.4 fmol (S.E.M.)/5 X 10(5) cells. Regulated insulin release was maintained through at least 15 passages in culture. The cells showed morphological evidence of senescence after passage 26 and this was associated with significant reduction in stimulated insulin release as well as insulin content. The ability of the cells of this clonal line to grow in soft agar suggests that it is a precursor cell line. The clonal B cell lines isolated so far may thus represent variably committed rather than fully differentiated B cells in culture. These clonal non-neoplastic cell lines will be useful models with which to study the regulation of maturation/differentiation of B cells and insulin gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of retinoids on the growth, ultrastructure, and cytoskeletal structures of malignant rat osteoblasts.

A clonal rat osteogenic sarcoma cell line, UMR 106-06, was used to study the effects of retinoic acid (RA) on its growth and morphology. Retinoic acid caused a reversible, time and dose-dependent inhibition of growth. RA-treated cells were larger, were more adherent to the substratum, and contained fewer mitotic figures. Half-maximal growth inhibition was observed at 10(-8) M. Among the naturally occurring retinoids, RA was clearly the most potent while the arotinoids, Ro 13-7410 and Ro 13-6298, were approximately 50 times more potent than was RA. A similar range of potencies was observed in the cloning efficiencies of the cells in soft agar. Fluorescence microscopy revealed that RA treatment increased the cellular content and organization of F-actin fibers. Ultrastructural changes include decreased chromatin dispersion and increased number of nucleoli per nucleus, decreased rough endoplasmic reticulum, decreased electron density and number of mitochondria, and increased formation of microfilaments and microtubules. These results identify this clonal cell line, which has been extensively characterized as the malignant counterpart of the normal osteoblast, as a target for vitamin A action.