J Korean Med Sci.  2004 Dec;19(6):853-858. 10.3346/jkms.2004.19.6.853.

Differential Expression of TGF-beta Isoforms During Differentiation of HaCaT Human Keratinocyte Cells: Implication for the Separate Role in Epidermal Differentiation

• Cho HR
• Hong SB
• Kim YI
• Lee JW
• Kim NI

• KMID: 1778568
• DOI: http://doi.org/10.3346/jkms.2004.19.6.853

Abstract

The three mammalian isoforms of transforming growth factor-beta (TGF-beta1, beta2, beta3) are potent regulators of cell growth, differentiation, and extracellular matrix deposition. To study their role in skin differentiation, we investigated the expression of TGF-beta isoforms on cell growth and differentiation induction of the human keratinocyte cell line, HaCaT by elevating the Ca2+ concentration. An ELISA and RT-PCR assay revealed secreted TGF-beta 1 protein and TGF-beta1 mRNA were increased during calci-um-induced differentiation. In contrast, major differences were seen for TGF-beta 2 and TGF-beta 3 mRNA which were decreased during differentiation, but TGF-beta 2 and TGF-3beta protein were not evident on an ELISA. These results suggest different functions for each TGF-beta isoforms in epidermal differentiation, such that TGF-beta 1 is associ-ated with the more differentiated state, and TGF-beta 2 and TGF-beta 3 may be associ-ated the more proliferated state.

Keyword

Transforming Growth Factor beta; Cell Differentiation; Keratinocytes; Calcium

MeSH Terms

Cell Differentiation/physiology
Cell Line
Gene Expression Regulation, Developmental/*physiology
Humans
Keratinocytes/*cytology/*physiology
Protein Isoforms/metabolism
Transforming Growth Factor beta/*metabolism

Figure

• 

  Fig. 1 TGF-β1 abundance are significantly increased at 0.1, 0.7, 1.0 and 1.2 mM Ca2+ compared with cells grown at 0.03 mM Ca2+. HaCaT keratinocytes were grown in the presence of increasing Ca2+ concentrations in the medium for 24 hr and 48 hr, respectively. TGF-β1 protein level in supernatant media was determined by ELISA assay (duplicates). Values are the mean±SD of 2 samples in 2 experiments. Graphical representation of TGF-β1 abundance in HaCaT conditioned medium. (n=2) *p<0.05 when compared with 0.03 mM Ca2+ condition.

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  Fig. 2 The mRNA expression of TGF-β1, TGF-β2, TGF-β3, and β-actin in HaCaT human keratinocyte cells. The mRNA levels of TGF-β isoforms and β-actin were analyzed by RT-PCR. PCR-products were separated on a 2% agarose gel and stained with ethidium bromide. Lane 1: TGF-β1, lane 2: TGF-β2, lane 3: TGF-β3, lane 4: β-actin.

• 

  Fig. 3 TGF-β1 mRNA levels are increased at 0.1, 0.7, 1.0 and 1.2 mM Ca2+ compared with cells grown at 0.03 mM Ca2+. RT-PCR of total RNA extracted from HaCaT keratinocytes exposed to increasing calcium concentration in the culture medium for 24 and 48 hr, respectively. (A) Representative RT-PCR showing TGF-β1 mRNA in all samples. (B) Graphical representation of combined densitometric analyses of TGF-β1 mRNA levels. Results are represented as the ratio of expression of TGF-β1 to that of β-actin. (n=3) *p<0.05 when compared with 0.03 mM Ca2+ condition.

• 

  Fig. 4 TGF-β2 mRNA levels are decreased at 0.1, 0.7, 1.0 and 1.2 mM Ca2+ compared with cells grown at 0.03 mM Ca2+. RT-PCR of total RNA extracted from HaCaT keratinocytes exposed to increasing calcium concentration in the culture medium for 24 and 48 hr, respectively. (A) Representative RT-PCR showing TGF-β2 mRNA in all samples. (B) Graphical representation of combined densitometric analyses of TGF-β2 mRNA levels. (n=3) *p<0.05 when compared with 0.03 mM Ca2+ condition.

• 

  Fig. 5 TGF-β3 mRNA levels are decreased at 0.1, 0.7, 1.0 and 1.2 mM Ca2+ compared with cells grown at 0.03 mM Ca2+. RT-PCR of total RNA extracted from HaCaT keratinocytes exposed to increasing calcium concentration in the culture medium for 24 and 48 hr, respectively. (A) Representative RT-PCR showing TGF-β3 mRNA in all samples. (B) Graphical representation of combined densitometric analyses of TGF-β3 mRNA levels. (n=3) *p<0.05 when compared with 0.03 mM Ca2+ condition.

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