Yonsei Med J.  2011 Jan;52(1):165-172. 10.3349/ymj.2011.52.1.165.

Duration and Magnitude of Extracellular Signal-Regulated Protein Kinase Phosphorylation Determine Adipogenesis or Osteogenesis in Human Bone Marrow-Derived Stem Cells

• Jung HS
• Kim YH
• Lee JW

• KMID: 1106452
• DOI: http://doi.org/10.3349/ymj.2011.52.1.165

Abstract

PURPOSE
Imbalances between osteogenic and adipogenic differentiation leads to diseases such as osteoporosis. The aim of our study was to demonstrate the differences in extracellular signal-regulated kinase (ERK) phosphorylation during both adipogenesis and osteogenesis of human bone marrow-derived stem cells (BMSCs).
MATERIALS AND METHODS
Using troglitazone, GW9662 and U0126, we investigated their role in hBMSC differentiation to adipogenic and osteogenic fates.
RESULTS
ERK1/2 inhibition by U0126 suppressed proliferator-activated receptor (PPAR)gamma expression and lipid accumulation, while it decreased the mRNA expression of adipogenic genes (lipoprotein lipase, PPARgamma, and adipocyte protein) and osteogenic genes (type I collagen and osteopontin). ERK phosphorylation was transient and decreased during adipogenesis, whereas it occurred steadily during osteogenesis. Troglitazone, a PPARgamma agonist, induced adipogenesis by inhibiting ERK phosphorylation even in an osteogenic medium, suggesting that ERK signaling needs to be shut off in order to proceed with adipose cell commitment. Cell proliferation was greatly increased in osteogenesis but was not changed during adipogenesis, indicating that ERK might play different roles in cellular proliferation and differentiation between the two committed cell types.
CONCLUSION
The duration and magnitude of ERK activation might be a crucial factor for the balance between adipogenesis and osteogenesis in human bone marrow-derived stem cells.

Keyword

Adipogenesis; osteogenesis; ERK; PPARgamma; human bone marrow-derived stem cells

MeSH Terms

Adipogenesis/*drug effects/genetics
Adult
Anilides/pharmacology
Bone Marrow Cells/*cytology/drug effects/metabolism
Butadienes/pharmacology
Cell Differentiation/drug effects
Cells, Cultured
Chromans/pharmacology
Extracellular Signal-Regulated MAP Kinases/*metabolism
Female
Humans
Male
Middle Aged
Nitriles/pharmacology
Osteogenesis/*drug effects/genetics
PPAR gamma/agonists/antagonists & inhibitors
Phosphorylation/drug effects
Reverse Transcriptase Polymerase Chain Reaction
Stem Cells/*cytology/drug effects/*metabolism
Thiazolidinediones/pharmacology

Figure

• 

  Fig. 1 Effect of agonists or antagonists of PPARγ and U0126 on adipogenic or osteogenic differentiation. (A) Human BMSCs were cultured in an adipogenic medium with or without troglitazone (25 µM), GW9662 (20 µM), or U0126 (20 µM) for 7 days. The lysates were subjected to western blot analysis. (B) BMSCs were induced adipocyte differentiation in the adipogenic medium (AD) for 14 days, and stained by Oil red O or Alizarin red S. (C) BMSCs were differentiated into osteoblasts in an osteogenic medium (OS) for 14 days, and stained by Oil red O or Alizarin red S. (D) The degree of adipogenic or osteogenic differentiation was determined as OD units by destaining (*p < 0.05, vs media). GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PPARγ, peroxisome proliferator-activated receptor; BMSCs, bone marrow-derived stem cells.

• 

  Fig. 2 ERK phosphorylation during the adipogenesis and osteogenesis of human BMSCs. (A) Human BMSCs were cultured in an adipogenic medium (AD) with or without troglitazone, GW9662, or U0126 for 5 days. The lysates were subjected to western blot analysis using antibodies specific for ERK1/2 and pERK. (B) Human BMSCs were cultured in an osteogenic medium (OS) with or without troglitazone, GW9662, or U0126. The lysates were subjected to western blot analysis using antibodies specific for ERK1/2 and pERK. ERK, extracellular signal-regulated kinase; BMSCs, bone marrow-derived stem cells.

• 

  Fig. 3 Change in total cell numbers during the osteogenesis and adipogenesis of human BMSCs. (A) Cells were incubated in an osteogenic (OS) (c and e) or adipogenic (AD) (d and f) medium for 14 days, and stained by Alizarind red S (a and c), Oil red O (b and d), and crystal violet (e and f). a and b are control comparing to adipogenesis and osteogenesis. a and b cultured control medium, DMEM-low glucose with 10% fetal bovine serum. (B) Crystal violet (CV) stain was quantified by destaining with 95% ethanol, and expressed as OD units (*p < 0.05, vs -OS media). BMSCs, bone marrow-derived stem cells.

• 

  Fig. 4 RT-PCR analysis during the adipogenesis (AD) and osteogenesis (OS) of BMSCs. (A) BMSCs were cultured in the adipogenic medium with or without troglitazone, GW9662, or U0126. Equal aliquots of the total RNA were reverse transcribed and amplified with the oligonucleotide primers specific for LPL, aP2, and PPARγ2. (B) Cells were induced with troglitazone compounds in the osteogenic medium for 7 days. Equal aliquots of total RNA were reverse transcribed and amplified with the oligonucleotide primers specific for LPL, aP2, PPARγ2, Type I collagen, and osteopontin. BMSCs, bone marrow-derived stem cells; LPL, lipoprotein lipase; aP2, adipocyte protein; PPARγ2, peroxisome proliferator-activated receptorγ2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

• 

  Fig. 5 pERK control of aidpogenesis and osteogenesis of BMSCs. ERK phosphorylation is involved in differentiation and proliferation during osteogenesis and adipogenesis. BMSCs, bone marrow-derived stem cells; ERK, extracellular signal-regulated kinase.

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