477 EFFECTS OF METHOTREXATE ON OSTEOELASTIC CELLS IN VITRO. G. Pioli, M. Pedrazzoni, G. Ferraccioli, G. Musetti, V. Ulietti, G, Rasini, M. Passeri. Istituto di Clinica %cdica Generale. Universita' di Parma. Italv.

Since 'methotrexate (MTX), an ant-ineoplastic and immunosuppressive agent, has been shown to favour the development of osteoporosis, we have examined the effects of the drug on osteoblastic cells in vitro.

We have used the murine osteogenic sarcoma cell line UMR 106.01, and human osteoblast-like cells grown from trabecular explants according to the method of Beresford et al. (1984). On both cell types we have measured the effects of MTX (1O-g to 1D-7 M) on cell proliferation and t:p;:ne phosphatese (ALP) a?d interleukin-6

production. Proliferation was quantitated by measuring, after solubilization, the uptake and metabolism of 3-(4,5-dimethyl- thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). IL-6 was measured on supernatants collected after 24 h of incubation, whereas p.L? was measured on cell lysates after 72 h. IL-6 was measured by a bioassay based on the proliferation of the murine hybridoma 89. ALP was determined by a spectrophotometric method at 405 nm, using p-nitrophenylphosphate as a substrate and corrected for protein content, as determined by the Bradford method.

The results of the study s ow that WTX, at the concentration of 5*10- $ and low7 M, suppresses osteoblast proliferation and ALP production, while IL-6 release is stimulated.

These findings indicate that MTX exerts direct effects on osteoblastic cells, which may explain its negative action on bone.

479 mRNA RXPRRSSION OF OSTEOBMSTIC WARRERS IN NOUL m BONE CELLS IN CULTURE. & Sutherland. L.C. Rao. S, pluzaffar. #J.Sodek. R.McBroome. *M.R.Haussler, J’.M.Murray. Dept. Medicine and #MRC Group in Periodon- tal Physiology, University of Toronto, Toronto, Canada and *Dept. Biochemistry, University of Arizona, Tucson, Arizona.

Previous studies in our laboratory have shown that human osteoblasts continuously exposed to dexamethasone from trabecular explant during cellular outgrowth from trabecular and subculture (HOB+DEX) exhibited a strik- ingly different phenotype from those cultured in the absence of dexamethasone (HOB-DEX) (Wong et al., 199@, J Bone Min Res 5: 803-813). To further characterize these 2 cell populations, Northern and slot blot analyses were used to examine the gene expressions of the osteoblastic markers alkaline phosphatase. collagen I and receptors for 1,25-dlhydroxyvitamin D (VDR) and estrogen (ER) , Cells derived from iliac crest bone bioosies were confluent 6 to 8 weeks after explant and verb used for RNA isolation (Chomcynski and Sacchi, 1987. Anal Biochem 162: 156-159) 7 to 10 days after subculture. We found that (HOB+Dwt) cells ixhibited statistically higher basal mRNA levels for alkaline phosphatase. collagen I and VDR when compared to levels expressed by (HOB-DRX) cells (p < 0.05). In (NOB-DEX) cells, the age and sex of the donor did not affect the expression of the osteoblastic markers. However, (HOB+DEX) cells derived from male donors demonstrated higher mRNA levels for alkaline phosphatase. collagen I and VDR compared to cells cultured from female donors. These results support the concept that (HOB+DI%) and (HOB-DEX) cells represent different stages of os- teoblast differentiation and suggest that sex-dependent differences in gene expression are associated with a more differentiated phenotype.

478

PROTEIN KINASE C STlM’ULATES JPHOSPHATF TRANCPCIRT IN QSTJZOBLASTS. wet. M. I

. Genetics Unit. and Hunnan Gene)

---_---.--_ . . . . . ..Y.Y...

Shriners Hospital and Depts. Surgery tics, McGill &dversin. Montreal. Ouebec. Canada.

We examined the effect of ph&ol 12-m&&ate 13-acetate (PMA), a potent activator of protein kinase C on sodium-dependent Pi uptake by cultured mouse osteoblasts.

Cells wen isolated from newborn calvaria by a non enzymatic method and grown in DMEM supplemented with 10% fetal calf serum and ascorbate. Confluent cells in secondary cultures were pretreated with phorbol esters or vehicle alone in DMEM supplemented with 0.1% BSA for various incubation periods. The initial r;ltr: of Pi up- take was then determined by incubatin 15 mM HEPES (pH 7.61,O.l mM H

cells in a medium containing

of either sodium or choline chloride $

d PO, (1 pCi/ml) and 143 mM or 4 min af 37T. Activation of

protein kinase C by PMA was assessed by measuring the enzyme activity in cytosol and membrane fractions of cells.

PMA caused a rapid increase in sodium-dependent Pi uptake which was observed 2 min after incubation and maximal within 10 min. This stimulatory effect of PMA was dosn:&pendent in the range of l@ to l@M and maximal (3 fold) at 10% (PMA: 234 f 9 pmol Pi& DNA. 4 mrin versus control: 80 f 2 pmol Pi& DNA. 4 min, n = 4, p * 0.001~~. This PMA effect on Pi uptake appeared specific for thz sctivo phorbol ester R: the inactive analog, 4a-phorbol dideca- noa&, hrd no effect, and selective for Pi since the sodium-dependent &nine uptike was not altered by PMA aatment. Kinetic analysis of PMA-indticed stimulation of sodium-dependent Pi transport indicated an incmased apparent Vmax with no significant change in rppntglt Km. In cells in which protein kinase C had been down-regulated by an overnight incubation with high doses of PMA, the phorbol ester no longer enhanced the Pi uptake indicating a critical role for protein kinase C in this response.

These results suggest that protein kinase C plays a role in the regulation of sodium-depenrrn: ?i transpon in osteoblasts.

PROTEIN RINASE C (F’KC) PIAYS A ROLB IN THE RIZUIATION OF PARATHYRDID liOl?MONE (T;H)*SFNSITI’.‘E ADENYLATE CYCIASE (AC) BY GlJimSINE-5’-(#‘,y-I&MI) TRIPHOSPHATg [Cpp(NH)p’ u pao. J.N. Wvlie. kpt of Medicine, University of Toionto and Cal&an Research Laboratory, Division of Endocrinology and Metabolism, St. Michael’s Hospital, Toronto, Ontario.

Qur previous studies showed that FKC modulates PI’& and forskolin-sensitive AC in ROS 17/2.8 cells (Calcif Tiss Int 45:354, 1989). We have now s&died further the mechanism whereby FKC modulates the Cpp(NH)p regulation of AC in permsabilired ROS 17/2.8 cells. Confluent cultures of ROS 17/2.8 cells were trypsinized to obtain cell suspensions, incubated with the activator of PKC, phorbol 12.myristate 13-acetate (FMA), and then penneabilized for 2 mins with 20 ug/ml saponin . The pennaabilized cells were centrifuged, then resuspended and incubated in buffer containing vehicle

O& PIH and/or GPP(NH)P.

[ ]-ATP AC acsivity wss measured using

as substrate and the [ HIcAMP datenincd by the Saloman method. Addition of exogenous CppNHp stimulated the basal and m-stimulated AC in permeabilized, burg not unp~rmeabilited ROS 17/2.8 cells. GPP(NH)P (10 to lo- M) stimulated both the basal and PTli-sensitive AC in a dose-dependent manner. PMA (100 ti) ad&d to the cells prior to permeabilization stimlated both basal and PI%sensitive AC in a time-dependent manner with _imal effect at 2 min: the stimulatory effect persisted up to 30 min. PMA changed the rate of enzyme activation (V_) by

GPP(F)P* but did not modify the affinity (ED50 - 3-4 x10- M) of basal and PTH-sensitive enzyme for Gpp(NH)p. On the other hand, dose-dependent stimulation by Fl’H at different concentrations of Gpp(NH)p showed that Cpp(NH)p increased 2.5 x lo- ptoVyg a;lO~~~;, :; s;t:l;d(::;

the V_ but had no effect on the ED 0 for PM. A study of the time course of the Gpp(NH)p ef ect ?n vehicle- and z Pm-treated permeabillzed cells confirmed rho PKC effect on the rate of activation of the enzyme. Our studies suggest that PKC plays an Important role in the guanylnucleotide regulation of basal and m-sensitive AC in osteoblasts.

195