Inhibition of Protein Kinase CK2 Prevents Adipogenic Differentiation of Mesenchymal Stem Cells Like C3H/10T1/2 Cells

Abstract

Protein kinase CK2 as a holoenzyme is composed of two catalytic α- or α'-subunits and two non-catalytic β-subunits. Knock-out experiments revealed that CK2α and CK2β are required for embryonic development. Little is known about the role of CK2 during differentiation of stem cells. Mesenchymal stem cells (MSCs) are multipotent cells which can be differentiated into adipocytes in vitro. Thus, MSCs and in particular C3H/10T1/2 cells are excellent tools to study a possible role of CK2 in adipogenesis. We found downregulation of the CK2 catalytic subunits as well as a decrease in CK2 kinase activity with progression of differentiation. Inhibition of CK2 using the potent inhibitor CX-4945 impeded differentiation of C3H/10T1/2 cells into adipocytes. The inhibited cells lacked the observed decrease in CK2 expression, but showed a constant expression of all three CK2 subunits. Furthermore, inhibition of CK2 resulted in decreased cell proliferation in the early differentiation phase. Analysis of the main signaling cascade revealed an elevated expression of C/EBPβ and C/EBPδ and reduced expression of the adipogenic master regulators C/EBPα and PPARγ2. Thus, CK2 seems to be implicated in the regulation of different steps early in the adipogenic differentiation of MSC.

Keywords: adipogenesis; kinase inhibitor; protein kinase; transcription factors.

Figure 1

C3H/10T1/2 cells were differentiated for 12 days using IBMX, dexamethasone and insulin. (a) Bright field images showing the differentiation status at day 12 of differentiation. Control cells (without differentiation mix) were compared to cells differentiated with the mix. Lipid droplets were stained red with Oil Red O. Magnification 200×; (b,e) Cells were harvested at given time points during differentiation and protein expression was detected with specific antibodies using a Western Blot approach. GAPDH was used as loading control; (c) Quantification of the protein expression of CK2α, CK2α’, and CK2β normalized to GAPDH; (d) CK2 activity during adipogenic differentiation of C3H/10T1/2 cells over time shown by the incorporation of [³²P]phosphate into the CK2 specific substrate peptide RRRDDDSDDD.

Figure 2

C3H/10T1/2 cells were differentiated in the presence of DMSO or CX-4945. (a) Proliferating C3H/10T1/2 cells were treated with 5, 10, 15, or 20 µM CX-4945 or DMSO as a control for 24 and 48 h. CK2 kinase activity was determined in an in vitro phosphorylation assay; (b) Cells were differentiated with differentiation mix containing DMSO or 15 µM CX-4945 and harvested at given time points. CK2 activity was determined in the protein extracts to confirm the inhibition over the complete differentiation process; (c) Differentiation status at day 12 of differentiation in cells treated with DMSO or 15 µM CX-4945. Lipid droplets were stained with Oil Red O and bright field images were recorded at 200× magnification; (d) Proliferation of DMSO- or 15 µM CX-4945-treated cells was determined at 0, 24, 48, and 72 h after the start of differentiation. Cell numbers were normalized to 0 h and are presented half-logarithmic; (e) Metabolic activity was examined in C3H/10T1/2 cells using MTT assay after differentiation of DMSO- or 15 µM CX-4945-treated cells for 24 or 48 h. Results were normalized to DMSO control values; (f) Differentiating cells were harvested 24 or 48 h after start of differentiation and cleavage of caspase-3 and PARP indicating apoptosis was examined using Western blot analysis −: untreated, D: DMSO-treated, CX: 15 µM CX-4945-treated, F: full length, Cl: cleavage product; (g) Western blot images of protein extracts from cells differentiated in the presence of DMSO (D) or 15 µM CX-4945 (CX) using specific antibodies for the CK2-subunits. GAPDH was used as loading control for all Western blots.

Figure 3

C3H/10T1/2 cells were differentiated in the presence of DMSO (D) or 15 µM CX-4945 (CX) and harvested at given time points during differentiation. Proteins were extracted and separated on a 12.5% SDS-polyacrylamide gel and transferred to a PVDF-membrane. C/EBPβ, C/EBPδ, C/EBPα, and PPARγ2 were detected with specific antibodies. GAPDH was used as a loading control.

Figure 4

Schematic outline of the observations on adipogenic differentiation after inhibition of CK2 with CX-4945.