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. 2020 Nov 4;12(21):21253-21272.
doi: 10.18632/aging.103848. Epub 2020 Nov 4.

Regulation of the regenerative activity of dental pulp stem cells from exfoliated deciduous teeth (SHED) of children by TGF-β1 is associated with ALK5/Smad2, TAK1, p38 and MEK/ERK signaling

Hsiao-Hua Chang  1 , Il-Ly Chen  1 , Yin-Lin Wang  1 , Mei-Chi Chang  2   3 , Yi-Ling Tsai  1 , Wen-Chien Lan  4 , Tong-Mei Wang  1 , Sin-Yuet Yeung  3 , Jiiang-Huei Jeng  1   5   6
Affiliations

Regulation of the regenerative activity of dental pulp stem cells from exfoliated deciduous teeth (SHED) of children by TGF-β1 is associated with ALK5/Smad2, TAK1, p38 and MEK/ERK signaling

Hsiao-Hua Chang et al. Aging (Albany NY). .

Abstract

Transforming growth factor-β1 (TGF-β1) regulates wound healing/regeneration and aging processes. Dental pulp stem cells from human exfoliated deciduous teeth (SHED) are cell sources for treatment of age-related disorders. We studied the effect of TGF-β1 on SHED and related signaling. SHED were treated with TGF-β1 with/without pretreatment/co-incubation by SB431542, U0126, 5Z-7-oxozeaenol or SB203580. Sircol collagen assay, 3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide (MTT) assay, alkaline phosphatase (ALP) assay, RT-PCR, western blotting and PathScan phospho-ELISA were used to measure the effects. We found that SHED expressed ALK1, ALK3, ALK5, TGF-RII, betaglycan and endoglin mRNA. TGF-β1 stimulated p-Smad2, p-TAK1, p-ERK, p-p38 and cyclooxygenase-2 (COX-2) protein expression. It enhanced proliferation and collagen content of SHED that were attenuated by SB431542, 5Z-7-oxozeaenol and SB203580, but not U0126. TGF-β1 (0.5-1 ng/ml) stimulated ALP of SHED, whereas 5-10 ng/ml TGF-β1 suppressed ALP. SB431542 reversed the effects of TGF-β1. However, 5Z-7-oxozeaenol, SB203580 and U0126 only reversed the stimulatory effect of TGF-β1 on ALP. Four inhibitors attenuated TGF-β1-induced COX-2 expression. TGF-β1-stimulated TIMP-1 and N-cadherin was inhibited by SB431542 and 5Z-7-oxozeaenol. These results indicate that TGF-β1 affects SHED by differential regulation of ALK5/Smad2/3, TAK1, p38 and MEK/ERK. TGF-β1 and SHED could potentially be used for tissue engineering/regeneration and treatment of age-related diseases.

Keywords: aging-related diseases; alkaline phosphatase; collagen; cyclooxygenase-2; differentiation.

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Conflict of interest statement

CONFLICTS OF INTEREST: All authors denied any conflict of interest for this submission.

Figures

Figure 1
Figure 1
The stem cell characteristics of SHED. (A) Expression of STRO-1 and (B) expression of CD146 in cultured SHED. One representative picture of flow cytometric analysis data was shown.
Figure 2
Figure 2
Expression of various TGF-β related receptors in SHED and the effect of TGF-β1 on the Smad2, TAK1, ERK1/2 and p38 phosphorylation of SHED. (A) SHED cells were cultured in DMEM with 10%FBS for 24 hours. Total RNA was isolated for RT-PCR analysis of TGF-β related receptors (ALK1, ALK3, ALK5, TGF-β1RII, betaglycan, endoglin) expression, (B) SHED were exposed to TGF-β1 for 0-120 min (as indicated on graphs). Cell lysates were prepared and proteins were used for analysis of p-Smad2 expression by PathScan phospho-ELISA (OD450, Mean ± SE). *Denotes statistically significant difference when compared with control. (C) SHED were exposed to TGF-β1 for 0-120 min. Cell lysates were prepared and proteins were used for western blotting analysis of p-Smad2, p-TAK1, p-ERK1/2, p-p38 and GAPDH (control) protein expression. One representative western blotting picture was shown.
Figure 3
Figure 3
Effect of TGF-β1 on the morphology of SHED. (A) Control SHED, exposure to (B) 0.1 ng/ml TGF-β1, (C) 0.5 ng/ml TGF-β1, (D) 1 ng/ml TGF-β1, (E) 5 ng/ml TGF-β1 and (F) 10 ng/ml TGF-β1 for 5 days. One representative morphologic picture of cells was shown.
Figure 4
Figure 4
Effect of TGF-β1 on the growth of SHED. (A) Number of viable SHED after exposure to TGF-β1 for 5 days as estimated by MTT assay. Results were expressed as cell viability (% of control, Mean ± SE). (B) Effect of SB431542 on the TGF-β1-induced growth of SHED as analyzed by MTT assay. (C) Effect of 5z-7oxozeaenol on the TGF-β1-induced growth of SHED as analyzed by MTT assay. (D) Effect of U0126 on the TGF-β1-induced growth of SHED as analyzed by MTT assay. (E) Effect of SB203580 on the TGF-β1-induced growth of SHED as analyzed by MTT assay. *Denotes statistically significant difference when compared to control, #denotes statistically significant difference when compared with TGF-β1 (5 ng/ml)-treated group.
Figure 5
Figure 5
Effect of TGF-β1 on COX-2 protein expression of cultured SHED. (A) COX-2 protein expression of SHED after exposure to TGF-β1 for 24 hours. (B) Effect of SB431542 on the TGF-β1-induced COX-2 expression of SHED. (C) Effect of 5z-7oxozeaenol on the TGF-β1-induced COX-2 expression of SHED. (D) Effect of U0126 on the TGF-β1-induced COX-2 expression of SHED. (E) Effect of SB203580 on the TGF-β1-induced COX-2 expression of SHED. One representative western blot picture was shown.
Figure 6
Figure 6
Effect of TGF-β1 on the collagen content of cultured SHED. (A) Collagen content of SHED after exposure to TGF-β1 for 5 days as measured by Sircol collagen assay. Results were expressed Mean ± SE. (B) Effect of SB431542 on the TGF-β1-induced increase in collagen content of SHED. (C) Effect of 5z-7oxozeaenol on the TGF-β1-induced increase in collagen content of SHED. (D) Effect of U0126 on the TGF-β1-induced increase in collagen content of SHED. (E) Effect of SB203580 on the TGF-β1-induced increase in collagen content of SHED. *Denotes statistically significant difference when compared to control, #denotes statistically significant difference when compared with TGF-β1 (5 ng/ml)-treated group.
Figure 7
Figure 7
Effect of TGF-β1 on the ALP activities of cultured SHED as analyzed by ALP staining. (A) ALP staining of SHED after exposure to TGF-β1 for 5 days. (B) Effect of SB431542 on the TGF-β1-induced increase or decrease in ALP activity of SHED. (C) Effect of 5z-7oxozeaenol on the TGF-β1-induced increase or decrease in ALP activity of SHED. (D) Effect of U0126 on the TGF-β1-induced increase or decrease in ALP activity of SHED. (E) Effect of SB203580 on the TGF-β1-induced increase or decrease in ALP activity of SHED. One representative ALP staining result was shown.
Figure 8
Figure 8
Effect of TGF-β1 on the ALP activities of cultured SHED as analyzed by ALP enzyme activity assay. (A) Quantitative ALP enzyme activity assay of SHED with/without exposure to TGF-β1 for 5 days. (B) Effect of SB431542 on the TGF-β1-induced increase or decrease in ALP activity of SHED. (C) Effect of 5z-7oxozeaenol on the TGF-β1-induced increase or decrease in ALP activity of SHED. (D) Effect of U0126 on the TGF-β1-induced increase or decrease in ALP activity of SHED, (E) Effect of SB203580 on the TGF-β1-induced increase or decrease in ALP activity of SHED. *Denotes statistically significant difference when compared with respective control group. #Denotes statistically significant difference when compared with respective TGF-β1-treated group.
Figure 9
Figure 9
Effect of TGF-β1 on TIMP-1 and N-cadherin protein expression of cultured SHED. (A) TIMP-1 protein expression of SHED after exposure to TGF-β1 for 24 hours. (B) Effect of SB431542 on the TGF-β1-induced TIMP-1 expression of SHED. (C) Effect of 5z-7oxozeaenol on the TGF-β1-induced TIMP-1 expression of SHED. (D) N-cadherin protein expression of SHED after exposure to TGF-β1 for 24 hours. (E) Effect of SB431542 on the TGF-β1-induced N-cadherin expression of SHED. (F) Effect of 5z-7oxozeaenol on the TGF-β1-induced N-cadherin expression of SHED. One representative western blot picture was shown.
Figure 10
Figure 10
TGF-β1 binds to TGF-β receptors of SHED to stimulate downstream signaling pathways such as ALK5/Smad2, TAK1, p38 and MEK/ERK to regulate growth, differentiation etc, can be potentially applied for treatment of aging-related diseases including dermal aging, osteoporosis, and pulp necrosis etc.
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