PKCη Regulates the TGFβ3-induced Chondevrepogenic Differentiation of Human Mesenchymal Stem Cell

Abstract

Transforming growth factor (TGF) family is well known to induce the chondevrepogenic differentiation of mesenchymal stem cells (MSC). However, the precise signal transduction pathways and underlying factors are not well known. Thus the present study aims to evaluate the possible role of C2 domain in the chondevrepogenic differentiation of human mesenchymal stem cells. To this end, 145 C2 domains in the adenovirus were individually transfected to hMSC, and morphological changes were examined. Among 145 C2 domains, C2 domain of protein kinase C eta (PKCη) was selected as a possible chondevrepogenic differentiation factor for hMSC. To confirm this possibility, we treated TGFβ3, a well known chondevrepogenic differentiation factor of hMSC, and examined the increased-expression of glycosaminoglycan (GAG), collagen type II (COL II) as well as PKCη using PT-PCR, immunocytochemistry and Western blot analysis. To further evaluation of C2 domain of PKCη, we examined morphological changes, expressions of GAG and COL II after transfection of PKCη -C2 domain in hMSC. Overexpression of PKCη-C2 domain induced morphological change and increased GAG and COL II expressions. The present results demonstrate that PKCη involves in the TGF-β3-induced chondevrepogenic differentiation of hMSC, and C2 domain of PKCη has important role in this process.

Keywords: C2-domain.; Chondevrepogenesis; Human mesenchymal stem cell; PKCη; TGF-β3.

Fig. 1

Morphological change of hMSCs 7 days after 145 C2 domain containing adenovirus library transfection. The origin of 12 red boxed C2 domain was listed in Table 1. Human MSCs at 80% confluence were transfected with 100 MOI of Adv-GFP or Adv-C2 domain for 2 h, and media were changed with fresh DMEM+10% FBS. Seven day after infection, cell morphology was examined using an inverted fluorescence microscope (IX81, Olympus, Japan) coupled to a CCD camera (Olympus DP71).

Fig. 2

Chondevrepogenic differentiation of hMSCs in a micromass culture. (A) Pictures of differentiating hMSC aggregates from micromass culture. Bar: 500 μm. (B) PNA staining of cultures treated with TGF-β3 demonstrates a change in cellular condensation. Bar: 500 μm. (C) MSCs were cultured as micromass and treated with TGF-β3 (10 ng/ml). Cultures were stained with Alcian blue after two days.

Fig. 3

C2 domain containing gene expressions during chondevrepogenic differentiation. During chondevrepogenic differentiation of hMSC, C2 domain containing gene expressions were changed by TGF-β3.

Fig. 4

PKCη gene expression during chondevrepogenic differentiation. Addition of TGF-β3 led to rapid expression of PKCη, which increased at day 2, and was sustained until day 7.

Fig. 5

PKCη protein expression during chondevrepogenic differentiation. Chondevrepogenesis was initiated by micromass culture, supplemented with TGF-β3 (10 ng/ml), the COL II, COL I, and PKCη expression was determined by Western blot analysis at day 3.

Fig. 6

Effect of PKCη-C2 domain on differentiation of hMSCs. (A) In a monolayer culture, Adv-PKCη-C2 domain infection led to morphological change at day 7 after transfection. Transfected cells were confirmed by GFP expression. Bar: 20 μm. (B) Chondevrepogenesis of hMSCs after PKCη-C2 domain infection. Transfected cells were cultured for 7 days in monolayer, chondevrepogenesis was quantified by measuring the absorbance of bound Alcian blue at 620 nm. (C) Effect of PKCη-C2 domain on chondevrepogenic differentiation. After PKCη-C2 domain transfection, chondevrepogenesis was initiated by micromass culture. Chondevrepogenesis was measured by Alcian blue staining at 4 days after induction. Bar: 500 μm. (D) Effect of PKCη-C2 domain on COL II expression. Transfected cells were cultured for 7 days in monolayer, the COL II, COL I, and PKCη expression was determined by Western blot analysis.

Fig. 7

Effect of PKCη-C2 domain on NHFB. In a monolayer culture, Adv-PKCη-C2 domain transfection increased COL II expression.