Korean J Physiol Pharmacol.  2014 Aug;18(4):289-296. 10.4196/kjpp.2014.18.4.289.

Effect of Phorbol 12-Myristate 13-Acetate on the Differentiation of Adipose-Derived Stromal Cells from Different Subcutaneous Adipose Tissue Depots

• Song JK
• Lee CH
• Hwang SM
• Joo BS
• Lee SY
• Jung JS

• KMID: 2285522
• DOI: http://doi.org/10.4196/kjpp.2014.18.4.289

Abstract

Human adipose-tissue-derived stromal cells (hADSCs) are abundant in adipose tissue and can differentiate into multi-lineage cell types, including adipocytes, osteoblasts, and chondrocytes. In order to define the optimal harvest site of adipose tissue harvest site, we solated hADSCs from different subcutaneous sites (upper abdomen, lower abdomen, and thigh) and compared their proliferation and potential to differentiate into adipocytes and osteoblasts. In addition, this study examined the effect of phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, on proliferation and differentiation of hADSCs to adipocytes or osteoblasts. hADSCs isolated from different subcutaneous depots have a similar growth rate. Fluorescence-activated cell sorting (FACS) analysis showed that the expression levels of CD73 and CD90 were similar between hADSCs from abdomen and thigh regions. However, the expression of CD105 was lower in hADSCs from the thigh than in those from the abdomen. Although the adipogenic differentiation potential of hADSCs from both tissue regions was similar, the osteogenic differentiation potential of hADSCs from the thigh was greater than that of hADSCs from the abdomen. Phorbol 12-myristate 13-acetate (PMA) treatment increased osteogenic differentiation and suppressed adipogenic differentiation of all hADSCs without affecting their growth rate and the treatment of Go6983, a general inhibitor of protein kinase C (PKC) blocked the PMA effect. These findings indicate that the thigh region might be a suitable source of hADSCs for bone regeneration and that the PKC signaling pathway may be involved in the adipogenic and osteogenic differentiation of hADSCs.

Keyword

Differentiation; hADSCs; PKC; pPMA; Proliferation

MeSH Terms

Abdomen
Adipocytes
Adipose Tissue
Bone Regeneration
Chondrocytes
Flow Cytometry
Humans
Osteoblasts
Protein Kinase C
Stromal Cells*
Subcutaneous Fat*
Thigh
Protein Kinase C

Figure

• 

  Fig. 1 Immunophenotypical characterization of ADSCs isolated from different subcutaneous tissues (UA, LA and T). (A) FACS analysis revealed positive for CD73, CD90 and CD105 and negative for CD31, CD34 and CD45. Back and red histograms represent control and specific antibody staining, respectively. (B) Histogram of CD73, CD90 and CD105 positive population in ADSCs isolated from different subcutaneous tissues (UA, LA and T). Data are expressed as mean±SD (n=3). *p<0.05.

• 

  Fig. 2 Proliferation rate of hADSCs from different subcutaneous tissues. Cells were cultured in CGM)with or without 10 nM PMA, AIM with or without 10 nM PMA, and OIM with or without 10 nM PMA. Cell proliferation was evaluated on days 4, 7, 10, and 14 of culture. Data are indicated as the mean±SD (n=7). a,bp<0.05, significantly different from the CGM.

• 

  Fig. 3 Osteogenic differentiation of hADSCs from different subcutaneous tissues. (A) Effect of PMA on osteogenic differentiation of hADSCs from LA. hADSCs were cultured in CGM or OIM with or without PMA (1, 10 and 100 nM) for 14 days and then stained with Alizarin red S. Quantitative data were obtained from destained Alizarin red S absorbance. Data are indicated as the mean±SD (n=3). *p<0.05, significantly different from OIM. (B) hADSCs from the UA, LA, and T regions were cultured in CGM or OIM with or without 10 nM PMA for 14 days and then stained with Alizarin red S. (C) Quantitative data obtained from destained Alizarin red S absorbance. (D) Quantitative analysis of the expression of osteogenesis-related genes. hADSCs from UA, LA, and T regions were treated with CGM or OIM with or without 10 nM PMA for 7 days. Total RNA was extracted and the expression levels of ALP and RUNX2 were analyzed by real-time PCR. White, black, and grey bars represent CGM, OIM, and OIM with 10 nM PMA, respectively. Data are expressed as mean±SD (n=9). *p<0.05.

• 

  Fig. 4 Adipogenic differentiation of hADSCs from different subcutaneous tissues. (A) hADSCs from UA, LA, and T regions were cultured in control growth medium (CGM) or adipogenic induction medium (AIM) with or without 10 nM phorbol 12- myristate 13- acetate (PMA) for 14 days then stained with Oil Red O. (B) Quantitative data obtained from lipid droplets. (C) Quantitative analysis of the expression of adipogenesis-related genes. hADSCs from UA, LA and T regions were treated with CGM or AIM with or without 10 nM PMA for 7 days. Total RNA was extracted and the expression of PPARG and AP2 was analyzed by real-time PCR. White, black, and grey bars represent CGM, AIM, and AIM with 10 nM PMA, respectively. Data are expressed as mean±SD (n=9). *p<0.05.

• 

  Fig. 5 The effect of PKC inhibitor, Go6983, on osteogenesis and adipogenesis of hADSCs by PMA. After pretreatment of hADSCs from thigh regions with Go6983 for 1 hour, cells were cultured in the OIM or AIM with or without 10 nM PMA. After 7 days of culture, total RNA was extracted and the expression levels of ALP, RUNX2 (A), PPAR and aP2 (B) were analyzed by real-time PCR. After 3 weeks of culture, the cells were stained with Alizarin red S staining for osteogenic differentiation (C) and Oil-red O staining for adipogenic differentiation (D). The degree of Alizarin red S for quantification of mineral deposition and Oil-Red O for quantification of lipid were measured at 450 nm and 500 nm with a 96-well plate reader, respectively. Data are expressed as mean±SD (n=6). a) CGM, b) OIM, c) OIM+PMA, d) OIM+ PMA+Go6983, e) CGM, f) AIM, g) AIM+PMA, and h) AIM+PMA+Go6983. *p<0.05 and **p<0.01.

Reference

1. Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science. 1997; 276:71–74. PMID: 9082988.

2. Choi JS, Leem JW, Lee KH, Kim SS, Suh-Kim H, Jung SJ, Kim UJ, Lee BH. Effects of human mesenchymal stem cell transplantation combined with polymer on functional recovery following spinal cord hemisection in rats. Korean J Physiol Pharmacol. 2012; 16:405–411. PMID: 23269903.

3. Norambuena GA, Khoury M, Jorgensen C. Mesenchymal stem cells in osteoarticular pediatric diseases: an update. Pediatr Res. 2012; 71:452–458. PMID: 22430381.

4. Pati S, Gerber MH, Menge TD, Wataha KA, Zhao Y, Baumgartner JA, Zhao J, Letourneau PA, Huby MP, Baer LA, Salsbury JR, Kozar RA, Wade CE, Walker PA, Dash PK, Cox CS Jr, Doursout MF, Holcomb JB. Bone marrow derived mesenchymal stem cells inhibit inflammation and preserve vascular endothelial integrity in the lungs after hemorrhagic shock. PLoS One. 2011; 6:e25171. PMID: 21980392.

5. Zuk PA, Zhu M, Ashjian P, De Ugarte DA, Huang JI, Mizuno H, Alfonso ZC, Fraser JK, Benhaim P, Hedrick MH. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell. 2002; 13:4279–4295. PMID: 12475952.

6. Konno M, Hamabe A, Hasegawa S, Ogawa H, Fukusumi T, Nishikawa S, Ohta K, Kano Y, Ozaki M, Noguchi Y, Sakai D, Kudoh T, Kawamoto K, Eguchi H, Satoh T, Tanemura M, Nagano H, Doki Y, Mori M, Ishii H. Adipose-derived mesenchymal stem cells and regenerative medicine. Dev Growth Differ. 2013; 55:309–318. PMID: 23452121.

7. Phinney SD, Stern JS, Burke KE, Tang AB, Miller G, Holman RT. Human subcutaneous adipose tissue shows site-specific differences in fatty acid composition. Am J Clin Nutr. 1994; 60:725–729. PMID: 7942579.

8. El-Mrakby HH, Milner RH. Bimodal distribution of the blood supply to lower abdominal fat: histological study of the microcirculation of the lower abdominal wall. Ann Plast Surg. 2003; 50:165–170. PMID: 12567054.

9. Misra A, Garg A, Abate N, Peshock RM, Stray-Gundersen J, Grundy SM. Relationship of anterior and posterior subcutaneous abdominal fat to insulin sensitivity in nondiabetic men. Obes Res. 1997; 5:93–99. PMID: 9112243.

10. Levi B, James AW, Glotzbach JP, Wan DC, Commons GW, Longaker MT. Depot-specific variation in the osteogenic and adipogenic potential of human adipose-derived stromal cells. Plast Reconstr Surg. 2010; 126:822–834. PMID: 20811215.

11. Schipper BM, Marra KG, Zhang W, Donnenberg AD, Rubin JP. Regional anatomic and age effects on cell function of human adipose-derived stem cells. Ann Plast Surg. 2008; 60:538–544. PMID: 18434829.

12. Clemens MJ, Trayner I, Menaya J. The role of protein kinase C isoenzymes in the regulation of cell proliferation and differentiation. J Cell Sci. 1992; 103:881–887. PMID: 1487501.

13. Gallicano GI, Yousef MC, Capco DG. PKC--a pivotal regulator of early development. Bioessays. 1997; 19:29–36. PMID: 9008415.

14. Kim EC, Lee MJ, Shin SY, Seol GH, Han SH, Yee J, Kim C, Min SS. Phorbol 12-Myristate 13-Acetate Enhances Long-Term Potentiation in the Hippocampus through Activation of Protein Kinase Cδ and ε. Korean J Physiol Pharmacol. 2013; 17:51–56. PMID: 23440225.

15. McCarty MF. Up-regulation of intracellular signalling pathways may play a central pathogenic role in hypertension, atherogenesis, insulin resistance, and cancer promotion--the 'PKC syndrome'. Med Hypotheses. 1996; 46:191–221. PMID: 8676754.

16. Nakura A, Higuchi C, Yoshida K, Yoshikawa H. PKCα suppresses osteoblastic differentiation. Bone. 2011; 48:476–484. PMID: 20951242.

17. Miraoui H, Oudina K, Petite H, Tanimoto Y, Moriyama K, Marie PJ. Fibroblast growth factor receptor 2 promotes osteogenic differentiation in mesenchymal cells via ERK1/2 and protein kinase C signaling. J Biol Chem. 2009; 284:4897–4904. PMID: 19117954.

18. Liu J, Someren E, Mentink A, Licht R, Dechering K, van Blitterswijk C, de Boer J. The effect of PKC activation and inhibition on osteogenic differentiation of human mesenchymal stem cells. J Tissue Eng Regen Med. 2010; 4:329–339. PMID: 20033927.

19. Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, Benhaim P, Lorenz HP, Hedrick MH. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng. 2001; 7:211–228. PMID: 11304456.

20. Stucki U, Schmid J, Hämmerle CF, Lang NP. Temporal and local appearance of alkaline phosphatase activity in early stages of guided bone regeneration. A descriptive histochemical study in humans. Clin Oral Implants Res. 2001; 12:121–127. PMID: 11251661.

21. Zou L, Zou X, Chen L, Li H, Mygind T, Kassem M, Bünger C. Multilineage differentiation of porcine bone marrow stromal cells associated with specific gene expression pattern. J Orthop Res. 2008; 26:56–64. PMID: 17676606.

22. Franceschi RT, Ge C, Xiao G, Roca H, Jiang D. Transcriptional regulation of osteoblasts. Cells Tissues Organs. 2009; 189:144–152. PMID: 18728356.

23. Peterman EE, Taormina P 2nd, Harvey M, Young LH. Gö 6983 exerts cardioprotective effects in myocardial ischemia/reperfusion. J Cardiovasc Pharmacol. 2004; 43:645–656. PMID: 15071351.

24. Liang D, Yang M, Guo B, Cao J, Yang L, Guo X. Zinc upregulates the expression of osteoprotegerin in mouse osteoblasts MC3T3-E1 through PKC/MAPK pathways. Biol Trace Elem Res. 2012; 146:340–348. PMID: 22081405.

25. Richardson JA, Amantea CM, Kianmahd B, Tetradis S, Lieberman JR, Hahn TJ, Parhami F. Oxysterol-induced osteoblastic differentiation of pluripotent mesenchymal cells is mediated through a PKC- and PKA-dependent pathway. J Cell Biochem. 2007; 100:1131–1145. PMID: 17031848.

26. Fleming I, MacKenzie SJ, Vernon RG, Anderson NG, Houslay MD, Kilgour E. Protein kinase C isoforms play differential roles in the regulation of adipocyte differentiation. Biochem J. 1998; 333:719–727. PMID: 9677333.

27. Webb PR, Doyle C, Anderson NG. Protein kinase C-epsilon promotes adipogenic commitment and is essential for terminal differentiation of 3T3-F442A preadipocytes. Cell Mol Life Sci. 2003; 60:1504–1512. PMID: 12943236.

28. Artemenko Y, Gagnon A, Aubin D, Sorisky A. Anti-adipogenic effect of PDGF is reversed by PKC inhibition. J Cell Physiol. 2005; 204:646–653. PMID: 15754337.

29. Zhou Y, Wang D, Li F, Shi J, Song J. Different roles of protein kinase C-betaI and -delta in the regulation of adipocyte differentiation. Int J Biochem Cell Biol. 2006; 38:2151–2163. PMID: 16950644.

30. Park E1, Patel AN. PKC-delta induces cardiomyogenic gene expression in human adipose-derived stem cells. Biochem Biophys Res Commun. 2010; 393:582–586. PMID: 20152816.