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

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-alpha is constitutively expressed in bone marrow cells independently of hematopoietic growth factors in cultures. PKC-gamma mRNA is undetectable. Similarly, the expression of PKC-beta 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-kappaB 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-kappaB 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.

Fig 1. PKC expression on mouse bone marrow-derived macrophage and granulocyte

Bone marrow non-adherent cells (5 × 10⁵/ml) were cultured in the presence of M-CSF (10 ng/ml) or G-CSF (5 ng/ml) for various period of time as indicated. Adherent cells from culture containing M-CSF (A, C) and non-adherent cell culture containing G-CSF (B, D) were harvested, counted, washed. 2 × 10⁵ cells were lysed with SDS sample buffer. Samples were subjected to SDS-PAGE and immunoblot analyses with individual antibodies against cPKC (pan), MARKS, c-fms and actin (A, B). In addition, cells were lysed with Trizol Reagent for total RNA isolation. One μg of total RNA was used to generate the cDNA using random primer and M-MLV reverse transcription enzyme followed by PCR amplification of the individual cDNA using specific primers with standard reaction procedure. RT-PCR products were analyzed by running through 1.5% agarose gel followed by 0.5% ethidium bromide staining (C, D). R: RAW264.7 mouse monocyte-macrophage cell line. NT: no template.

Fig 2. PKC degradation on mouse bone marrow-derived macrophage by calpain and proteasome

Bone marrow non-adherent cells (5 × 10⁵/ml) were cultured in the presence of M-CSF (10 ng/ml) for various periods. Then, adherent cells were harvested, counted, washed and lysed with SDS sample buffer. Samples were loaded and run the SDS-PAGE followed by immunoblot analyses with individual antibodies against calpain, 20 s proteasome and actin (A). At day 7 of primary Cultures of bone marrow adherent cells, PKC expression level was evaluated by immunoblot assay with anti-PKC (pan) antibody after adding Alln (final concentration 10 μM) (B) or MG-132 (C) to the culture system for different time course.

Fig 3. Activation of NF-κB on bone marrow-derived macrophages and granulocytes

Freshly obtained mouse (A) and human (B) bone marrow non-adherent cells (5 × 10⁵/ml) were cultured in the presence of M-CSF (10 ng/ml) or G-CSF (5 ng/ml) for three days. After starvation in cytokine-free medium overnight, both adherent macrophage and non-adherent granulocytes were then re-exposed to M-CSF (10 ng/ml) or G-CSF (5 ng/ml) for indicated time periods. The cells were then lysed and nuclear extracts were isolated, followed by a mobility-shift assay with ATP-γ-³²P labeled NF-κB consensus sequence probe. Total nuclear protein (5 μg) was used per lane. (C) Sample was pre-mixed with either synthetic NF-κB binding oligo or mutant NF-κB binding oligo prior to EMSA for NF-κB activity.