Stimulation of prostaglandin E2 synthesis in cloned osteoblastic cells of mouse (MC3T3-E1) by epidermal growth factor.

K Yokota,M Kusaka,T Ohshima,S Yamamoto,N Kurihara,T Yoshino,M Kumegawa

doi:10.1016/s0021-9258(18)66726-2

Abstract

Prostaglandin (PG) E2, known as a bone-resorption factor, was released as a predominant arachidonate metabolite in the culture medium of an osteoblastic cell line cloned from mouse calvaria (MC3T3-E1). Epidermal growth factor (EGF) (10 ng/ml) prominently enhanced endogenous PGE2 synthesis, requiring the simultaneous presence of unidentified factor(s) contained in bovine serum. PGE2 synthesis increased after a lag phase for 1-2 h and reached a maximum level at about 3 h after EGF addition. EGF-stimulated PGE2 synthesis was almost completely blocked by 10 microM cycloheximide or 1 microM actinomycin D. Furthermore, when the cells were pretreated with EGF, the microsomes exhibited an increased activity of fatty acid cyclooxygenase (arachidonic acid----PGH2), whereas the activity of PGE synthase (PGH2----PGE2) remained unchanged. These results suggested an EGF-mediated induction of cyclooxygenase. Following increased PGE2 synthesis, DNA synthesis increased and alkaline phosphatase activity decreased in a slower response to EGF. PGE2 (above 0.1 microM) added to the cells could replace EGF. However, such effects of EGF on the osteoblasts could not be attributed totally to an autocrine function of PGE2 produced by stimulation with EGF because these effects of EGF were not abolished by indomethacin, which blocked the PGE2 synthesis.

Highlights

PGE2synthesis, DNA synthesis increased and alkaline phosphataseactivity decreased in a slower responstoe epidermal growth factor (EGF).PGE, addedtothe cells could
Endogenous PGE, Synthesis-The culture medium of MC3T3-El cells was extracted by the use of a Sep-Pak Cls replace EGF
Stimulation of Endogenous PGE, Synthesis by EGF-Severa1compounds which are known to regulate calcium metabolism in bone were added to MC3T3-El cells, and the PGEz released in the culture medium was determined by radioimmunoassay

Summary

RESULTS

PGE2synthesis, DNA synthesis increased and alkaline phosphataseactivity decreased in a slower responstoe EGF.PGE, (above 0.1 PM) addedtothe cells could. Endogenous PGE, Synthesis-The culture medium of MC3T3-El cells was extracted by the use of a Sep-Pak Cls replace EGF. The bone tissue is composed of various types of cell including osteoblasts and osteoclasts. It had not been clarified until recently which type of cell was responsible for the biosynthesis of PGE,. A cloned osteosarcoma cell line with osteoblastic features produced PGE, (7), and a primary culture of osteoblast-like cells from chick embryo calvaria produced various amount was ruled out by using another solvent system for reverse-phase HPLC. PGE, was detected as a major product. The Sep-Pak CIS extract was fractionated by reverse-phase HPLC,and each fraction was subjected to radioimmunoassay of PGE,.

Present address

O 2 0 30

DISCUSSION