Abstract
It is generally accepted that the multiple, similar protein kinase C (PKC) isozymes are responsible for different specialized physiological processes, but evidence that directly assigns specific functions to specific isozymes is scarce. To test whether specific PKC isozymes are involved in myeloid differentiation, we have studied the effect of overexpression of PKC-alpha, -beta II, -delta, -epsilon, -zeta and -eta in 32D, a mouse myeloid progenitor cell line that does not differentiate in response to 12-O-tetradecanoylphorbol-13-acetate (TPA). No significant morphological or phenotypic changes could be observed in unstimulated cells that overexpress any of these isozymes. However, the cell lines that overexpressed PKC-alpha or -delta had acquired the ability to become mature macrophages 2-6 h after TPA stimulation. The overexpression of PKC-beta II, -epsilon, -zeta, or -eta, in contrast, did not permit TPA-induced differentiation. These results indicate that only these two members of the PKC gene family can participate in TPA-induced myeloid differentiation.
Highlights
To test whether specific PKC iso- a major role in the acquisition of differentiation competence zymes are involved in myeloid differentiation, we have studied the effect of overexpression of PKC-a, -011, -6, - 6, -( and -q in 32D, a mouse myeloid progenitor cell line that does not differentiate in response to 12-0tetradecanoylphorbol-13-acetate (TPA)
E.g. HL-60(re- shown that prolonged activation of PKC by TPA is necessary viewed by Collins, 1987) or U937 (Ways et al, 1987), are able for HL-60 differentiation (Aiharaet al., 1991), no insight has to differentiate into mature macrophagesupon stimulation been obtained into whether specific isozymes of this family withphorbol esters,suchas 12-O-tetradecanoylphorbol-13- are involved inthis process
PKC isozymes in 32D cells, which enabled us to test whether overexpression of any of them could restore the ability to undergo TPA-induced macrophage differentiationseen in normal mouse myeloid progenitors
Summary
Vol 268, No 27, Issue of September 25, pp. 20110-20115, 1993 Printed in U.S.A. Harald MischakS8,Jacalyn H. Our studies indicated that most mouse myeloid cell overexpression of PKC-BII,-6, -f, or -q, in contrast, did lines express only low or undetectable levels of PKC-a and not permitTPA-induced differentiation. Seven of the TPA, but it is capable of differentiation into mature macroknown members of the PKC gene family are differentially phages if transfected with CSF-1-receptor and subsequently expressed in mouse hemopoietic cells (Mischak et al, 1991b). Lysozyme activity was assayed as described (Osserman and Lawlor, 1966).The ability of wildtype 32Dcells and the PKC overexpressers to phagocytize either before or after 16-h stimulation with 10 ng/ml TPA was determined by incubating IO7 myeloid cells with lo yeast particles in 10 mlof medium for 1 h at 37 "C. The PKC activity is expressed as counts/min of 32P incorporated into the substrate/pg protein of the partially purified lysate
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