Hypoxia-inducible factor-1α activates transforming growth factor-β1/Smad signaling and increases collagen deposition in dermal fibroblasts

Abstract

Hypoxia of local tissue occurs during the scar formation; however, the degree of ischemia and hypoxia in the central areas of keloids is more serious than those in normal scars. Hypoxia-induced factor (HIF), is one of the main cellular responses to hypoxia, allowing cells to adapt to low-oxygen conditions. We investigated the correlation among hypoxia, transforming growth factor-β1/Smad signaling and collagen deposition. Hypoxia up-regulated TGF-β1, Smad2/3, p-Smad2/3, Smad4, and total collagen in both normal and keloid fibroblasts via HIF-1α, which was attenuated by HIF-1α inhibition, but TβRII levels were not significantly altered. Silencing Smad4 under hypoxia decreased the mRNA and protein levels of HIF-1α, suggesting up-regulated Smad4 may also plays a role in promoting HIF-1α. Finally, we examined the role of the TGF-β1/Smad pathway in collagen deposition. When TβRII was inhibited by ITD-1 under hypoxic conditions, p-Smad2/3 levels and collagen deposition decreased. When inhibited TβRII by siRNA under normoxia, the levels of p-Smad2/3, Smad4 and collagen deposition also decreased. This result demonstrated that hypoxia promoted TGF-β1/Smad signaling via HIF-1α and that both HIF-1α and the TGF-β1/Smad signaling promotes collagen deposition in hypoxia, which is an important mechanism of keloid formation.

Keywords: collagen; hypoxia; hypoxia-Inducible factor-1α; keloid; transforming growth factor-β1/Smad.

Figure 1. Hypoxia promoted TGF-β/Smad signaling in HFFs and HKFs

(A) HIF-1α, TGF-β1, VEGF and CTGF protein levels were up-regulated by 24 h of hypoxia (1% O₂) exposure, but TβRII expression did not obviously differ between the normoxia and hypoxia groups. (B) ELISA was used to detect secreted TGF-β1 in serum-free medium after 6 h, 12 h and 18 h of exposure to normoxia (21% O₂) or hypoxia (1% O₂). (C, D) Quantitative reverse transcriptase-PCR (qRT-PCR) analyses of Smad2, Smad3, Smad4 and TGF-β1 mRNA levels in HFFs and HKFs after 24 h of hypoxia or normoxia exposure. Bars show the means±SE of three independent experiments (n = 3); ^*represents P < 0.05. (E) Western blotting shows the protein levels of HIF-1α, Smad2/3, p-Smad2/3 and Smad4 after 24 h of hypoxia or normoxia exposure. The histogram shows the protein band intensity ratio of p-Smad2 to Smad2 and p-Smad3 to Smad3. (F) The expression of HIF-1α and Smad2/3 was tested using immunohistochemistry analysis.

Figure 2. HIF-1α, Smad2/3, p-Smad2/3 and Smad4 was enhanced following treatment with 1% hypoxia

The protein expression and intracellular localization of HIF-1α, Smad2/3, p-Smad2/3 and Smad4 were detected by immunofluorescence staining in HFFs under normoxia or 24 h of hypoxia (1% O₂). HIF-1α and Smad4 localized mainly in the nucleus and Smad2/3 mainly in cytoplasm.

Figure 3. siHIF-1α inhibited TGF-β/Smad signaling in HFFs and HKFs

(A) qRT-PCR showing clear knockdown of HIF-1α by siHIF-1α transfection for 48 h. (B, D) 48 h after transfection, HFFs and HKFs were transferred to a 1% O₂ hypoxia incubator for 24 h, and siHIF-1α inhibited HIF-1α, TGF-β1, Smad4, Smad2/3, p-Smad2/3, CTGF and VEGF levels. The histogram shows the protein band intensity ratio of p-Smad2 to Smad2 and p-Smad3 to Smad3. (C) Before transferring cells to the hypoxia incubator, we replaced the culture media with serum-free media, and both the siHIF-1α and NC groups were treated with 1% O₂ for 12 h. The level of secreted TGF-β1 in the serum-free media was then measured.

Figure 4. siSmad4 inhibits HIF-1α in hypoxia

(A) qRT-PCR showing clear knockdown of Smad4 by siSmad4 transfection for 48 h. (B) 24 h after transfection, HFFs and HKFs were treated with 1% hypoxia or normoxia for 24 h. 48 h after transfection, the expression of mRNA level of HIF-1α was down-regulated in the group silencing Smad4. (C) After 72 h transfected by si-Smad4 and 24 h hypoxia treatment, the protein level of HIF-1α was down-regulated in the si-Smad4 group compared with the negative control group. The histogram shows the protein band intensity ratio of HIF-1α to β-Tubulin.

Figure 5. Total collagen deposition was promoted by acute hypoxia via HIF-1α and the TGF-β signaling pathway

(A) The ratio of deposited collagen to the total protein concentration was elevated after 24 h, 48 h, 72 h of hypoxia in both HFFs and HKFs. (B) At 48 h after transfection, HFFs and HKFs were transferred to a 1% O₂ hypoxia incubator for 24 h. Collagen deposition was clearly inhibited in the siHIF-1α group compared with the NC group. (C) Masson staining for collagen fiber (blue) of normal and keloid tissues. (D) Treatment with 5 μM ITD-1(dissolved in DMSO), DMSO, and blank, respectively, for 24 h under 1% O₂ condition. ITD-1 inhibited the phosphorylation of Smad2/3 by inhibiting TβRII. Meanwhile, HIF-1α and Smad2/3 levels remained stable in all three groups. The histogram shows the protein band intensity ratio of p-Smad2 to Smad2 and p-Smad3 to Smad3. (E) Collagen deposition was reduced in the ITD-1-treated HFFs and HKFs. (F) siTβRII reduced the protein levels of TβRII, p-Smad2/3 at 72 h after transfection in normoxia. HIF-1α protein levels remained stable in the two groups. The histogram shows the protein band intensity ratio of p-Smad2 to Smad2 and p-Smad3 to Smad3. (G) Collagen deposition was clearly inhibited in the siTβRII group compared with the NC group.