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Differentiation-Associated Expression of Conventional Protein Kinase C Isoforms in Primary Cultures of Bone Marrow Cells Induced by M-CSF and G-CSF

Barbara Ann Karmanos Cancer Institute, and Department of Internal Medicine, Wayne State University School of Medicine, Detroit, Michigan 48201

*Corresponding author:

Xiaohua Li, Ph.D.

9207 Scott Hall, 540 E. Canfield, Detroit, MI 48201

Tel.: 313-993-4188 

Fax: 313-577-0057 . 

E-mail address:  xhli@med.wayne.edu

Abstract

The Protein kinase C (PKC) -associated signal pathway plays crucial roles in regulation of cell growth, differentiation and apoptosis. The present study focuses on conventional PKC (cPKC) expression and its regulation in primary cultures of bone marrow cells induced to undergo macrophage/granulocyte differentiation by macrophage colony-stimulating factor (M-CSF) or granular colony-stimulating factor (G-CSF). By performing western blot analysis with pan anti-PKC antibodies, we found that PKC is transiently induced by M-CSF, reaching a maximum level by day 2, and then declines and diminishes by day 9 in primary culture of bone marrow cells.  In contrast, the expression of PKC along G-CSF induced granulocytic differentiation of bone marrow stem cells is low and increases gradually. Reverse transcription-PCR (RT-PCR) assay was utilized to investigate the expression of PKC isoforms. PKC-a is constitutively expressed in bone marrow cells independently of hematopoietic growth factors in cultures. PKC-γ mRNA is undetectable. Similarly, the expression of PKC-β is transiently induced by M-CSF, yet steadily increased by G-CSF, in agreement with results obtained from PKC protein expression. Furthermore, gel-shift assay showed that the activation of NF-kB is transiently induced by M-CSF but not by G-CSF.  These data suggest that PKC expression is involved in both macrophage and granulocyte differentiation by bone marrow committed stem cells. Yet, NF-kB activation is only detected in macrophage and not granulocyte differentiation.  Thus, we conclude that the PKC-mediated signaling pathway is distinctly involved in bone-marrow cell differentiation induced by M-CSF and G-CSF.

Keywords: bone marrow; macrophage/granulocytes differentiation; NF-kB; PKC

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