Korean J Physiol Pharmacol.  2016 Sep;20(5):459-466. 10.4196/kjpp.2016.20.5.459.

Lnk is an important modulator of insulin-like growth factor-1/Akt/peroxisome proliferator-activated receptor-gamma axis during adipogenesis of mesenchymal stem cells

• Lee JH
• Lee SH
• Lee HS
• Ji ST
• Jung SY
• Kim JH
• Bae SS
• Kwon SM

• KMID: 2350502
• DOI: http://doi.org/10.4196/kjpp.2016.20.5.459

Abstract

Adipogenic differentiation of mesenchymal stem cells (MSCs) is critical for metabolic homeostasis and nutrient signaling during development. However, limited information is available on the pivotal modulators of adipogenic differentiation of MSCs. Adaptor protein Lnk (Src homology 2B3 [SH2B3]), which belongs to a family of SH2-containing proteins, modulates the bioactivities of different stem cells, including hematopoietic stem cells and endothelial progenitor cells. In this study, we investigated whether an interaction between insulin-like growth factor-1 receptor (IGF-1R) and Lnk regulated IGF-1-induced adipogenic differentiation of MSCs. We found that wild-type MSCs showed greater adipogenic differentiation potential than Lnk(-/-) MSCs. An ex vivo adipogenic differentiation assay showed that Lnk(-/-) MSCs had decreased adipogenic differentiation potential compared with wild-type MSCs. Interestingly, we found that Lnk formed a complex with IGF-1R and that IGF-1 induced the dissociation of this complex. In addition, we observed that IGF-1-induced increase in the phosphorylation of Akt and mammalian target of rapamycin was triggered by the dissociation of the IGF-1R-Lnk complex. Expression levels of a pivotal transcription factor peroxisome proliferator-activated receptor gamma (PPAR-γ) and its adipogenic target genes (LPL and FABP4) significantly decreased in Lnk(-/-) MSCs. These results suggested that Lnk adaptor protein regulated the adipogenesis of MSCs through the IGF-1/Akt/PPAR-γ pathway.

Keyword

Adipogenesis; Insulin-like growth factor-1; Lnk; Mesenchymal stem cell; Peroxisome proliferator-activated; receptors gamma

MeSH Terms

Adipogenesis*
Endothelial Progenitor Cells
Hematopoietic Stem Cells
Homeostasis
Humans
Insulin-Like Growth Factor I
Mesenchymal Stromal Cells*
Phosphorylation
PPAR gamma
Sirolimus
Stem Cells
Transcription Factors
Insulin-Like Growth Factor I
PPAR gamma
Sirolimus
Transcription Factors

Figure

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  Fig. 1 Differentiation of adipose tissue-derived mesenchymal stem cells.(A) Wild-type (WT) and Lnk–/– mesenchymal stem cells (MSCs) were differentiated in vitro into chondrocytes, osteocytes, and adipocytes. (B) Osteocyte differentiation was quantified as the relative Alizarin red S stained area. Values represent the mean±standard error of mean (SEM). (C) Adipocyte differentiation was quantified as the number of oil red O stained cells. Values represent the mean±SEM; **p<0.01 vs. WT MSC. (D) Chondrocyte differentiation was quantified as the number of collagen II stained cells. Values represent the mean±SEM. (E) Lnk mRNA expression during adipocyte differentiation in wild-type MSCs was measured by performing quantitative reverse transcription-polymerase chain reaction (RT-PCR). Values are expressed as mean±SEM; **p<0.01 vs. values obtained after 4 days.

• 

  Fig. 2 In vivo adipogenic differentiation of adipose tissue-derived MSCs.(A) Wild-type (WT) or Lnk–/– MSCs were labeled with PKH-67 (green fluorescence), injected subcutaneously into nude mice, and allowed to differentiate for 10 days. Tissue sections showing green fluorescence were stained with hematoxylin or oil red O. Dotted lines indicate PKH-67- or oil red O-stained sites. (B) Adipocyte differentiation was quantified as the relative oil red O stained area. Values represent the mean±standard error of mean (SEM); **p<0.01 vs. WT MSC.

• 

  Fig. 3 Insulin-like growth factor-1-mediated dissociation of the insulinlike growth factor-1 receptor–Lnk complex promotes the phosphorylation of Akt/mTOR.(A) Wild-type MSCs were treated with insulin-like growth factor-1 (IGF-1) for 10 min. Coimmunoprecipitation was performed using antibodies against IGF-1 receptor (IGF-1R) and Lnk. (B and C) Wild-type MSCs were treated with IGF-1 for 10 min, and formation of phosphorylated Akt or mTOR was detected by performing western blotting. The lower panels denote the mean±SEM for each condition, as determined from densitometry relative to β-actin; **p<0.01 vs. wild-type MSCs not treated with IGF-1, ##p<0.01 vs. Lnk–/– MSCs treated with IGF-1.

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  Fig. 4 Involvement of Akt and mTOR signaling in IGF-1-induced differentiation of MSCs.(A) Wild-type (WT) or Lnk–/– MSCs were treated with Akt inhibitor (10 nM) and rapamycin (1 nM). Adipocyte differentiation was induced by incubating the cells in an IGF-1-containing differentiation medium for 14 days and was assessed by staining with oil red O. (B) Adipocyte differentiation was quantified as the number of oil red O stained cells. Values represent the mean±standard error of mean (SEM); **p<0.01 vs. control WT MSC, #p<0.05 and ##p<0.01 vs. control Lnk–/– MSC, ¶¶p<0.01 vs. IGF-treated WT MSC, §§p<0.01 vs. IGF-treated Lnk–/– MSC, $$p<0.01 vs. Lnk–/– MSC treated with PBS (Control), IGF-1 (IGF-1), IGF pretreated with Akt inhibitor (IGF-1+Akt inhibitor), or IGF pretreated Rapamycin (IGF-1+Rapamycin), respectively.

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  Fig. 5 Expression of PPAR-γ, LPL, and FABP4 during adipogenic differentiation.(A) Adipogenic differentiation of wild-type or Lnk–/– MSCs was induced by incubating the cells in an adipogenic differentiation medium for 10 days. PPAR-γ level was determined by performing western blotting. Bar graph denotes mean±SEM, as determined from densitometry relative to β-actin; **p<0.01 vs. wild-type MSCs (0 day), ##p<0.01 vs. Lnk–/– MSCs (10 day). (B and C) Wild-type or Lnk–/– MSCs were treated with or without 2 ng/ml IGF-1 for 4 days. LPL and FABP4 mRNA expression was quantified by performing quantitative RT-PCR. Values are expressed as mean±SEM; **p<0.01 vs. wild-type MSCs not treated with IGF-1, ##p<0.01 vs. Lnk–/– MSCs treated with IGF-1.

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  Fig. 6 Adipogenesis and PPAR-γ expression in Lnk–/– MSCs after overexpression of Akt.Akt overexpression in Lnk–/– MSCs were performed by retrovirus-mediated expression of Akt. (A) Adipocyte differentiation was induced by incubating normal or Akt overexpressed Lnk–/– MSCs in an IGF-1-containing differentiation medium for 14 days and was assessed by staining with oil red O. (B) Adipogenic differentiation of normal or Akt overexpressed Lnk–/– MSCs was induced by incubating the cells in an adipogenic differentiation medium for 10 days. PPAR-γ level was determined by performing western blotting.

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