Human adult dermal fibroblasts (lot no. 61447289) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and seeded at a density of 10,000 cells/cm² in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% antibiotic-antimycotic in a humidified incubator at 37°C with 5% CO₂. To induce hypoxia, the cells were placed in three-gas incubator (Thermo Fisher Scientific, Inc., Waltham, MA, USA) that maintains a sub-ambient O₂ level (1%, 5% or 10%) with or without 10 ng/ml of TGF-β1 (Peprotech, Rocky Hill, NJ, USA) by the regulated injection of N₂ for 48 h. The control cells were placed in a similar incubator which was maintained at 5% CO₂ and 21% oxygen level. All reagents were purchased from Invitrogen (Carlsbad, CA, USA) unless otherwise stated.

The cells were pre-incubated in PBS and fixed with 4% formaldehyde for 30 min, followed by incubation with rabbit anti-human p-Smad3 (9520; Cell Signaling Technology, Inc., Danvers, MA, USA) overnight at 4°C. After washing with PBS, goat anti-rabbit IgG-CFL 555 secondary antibody (sc-362272; Santa Cruz Biotechnology, Santa Cruz, CA, USA) was added in 1% BSA followed by incubation for 1 h at room temperature in the dark. Images were obtained using a FSX100 microscope (Olympus Corp., Tokyo, Japan). Nuclei were counterstained using DAPI (Sigma-Aldrich, St. Louis, MO, USA).

Flow cytometry (BD FACSAria; BD Biosciences, Franklin Lakes, NJ, USA) was performed to detect cell apoptosis. The following 2 groups were under investigation: i) the control group and ii) the 1% oxygen group. We observed the apoptotic rates at 48 h post-treatment. In accordance with the Annexin V/propidium iodide (PI) apoptosis kit (BioVision, San Francisco, CA, USA), 5×10⁵ cells were collected in each tube and 1 ml Annexin V binding buffer was added followed by thorough mixing. Subsequently, 5 µl Annexin V-fluorescein isothiocyanate and 10 µl PI were added. After mixing, the tube was incubated in the dark at 37°C for 15 min. For the early apoptotic cells, membrane phosphatidylserine was exposed and combined with Annexin V but no PI. For the late apoptotic cells, the membranes were permeable to PI and the cells were stained with Annexin V and PI. The dead cells were stained only with PI.

The TRIzol reagent kit (Invitrogen) was used for RNA extraction. The isolated RNA was reverse transcribed into complementary DNA using the PrimeScript RT Reagent kit (Takara Biotechnology Co., Ltd., Dalian, China). Primers were obtained from Beijing AuGCT DNA-SYN Biotechnology Co., Ltd., (Beijing, China). Quantitative PCR (qPCR) was performed using the iQ5 real-time PCR detection system (Bio-Rad Laboratories, Inc., Hercules, CA, USA), using SYBR Premix Ex Taq II (obtained from Takara Biotechnology Co., Ltd.) in a 20 ml volume of the PCR reaction solution. The sequences for primers are listed as follows: HIF-1α forward, 5′-AGCCGAGGAAGAACTATGAAC-3′ and reverse, 5′-ATTTGATGGGTGAGGAATGGG-3′; and GAPDH forward, 5′-GCACCGTCAAGGCTGAGAAC-3′ and reverse, 5′-TGGTGAAGACGCCAGTGGA-3′. The results were normalized against the mean Ct-values of GAPDH using the ΔCt method as follows: ΔCt = C_{tgene of interest} − C_{tmean (GAPDH)}. The fold increase was calculated as 2^−ΔΔCt.

Total protein lysates were generated using RIPA lysis buffer supplemented with protease and phosphatase inhibitor mixtures (KC-440; Shanghai KangChen Biological Technology Co., Shanghai, China). Nuclear protein extracts were obtained using the NE-PER nuclear and cytoplasmic extraction reagents (Pierce Biotechnology, Inc., Rockford, IL, USA), according to the manufacturer's instructions. Proteins (40 µg) were loaded onto a 5–10% polyacrylamide gel, separated by electrophoresis and transferred onto a polyvinylidene difluoride (PVDF) membrane. After blocking with 5% non-fat milk, the PVDF membrane was incubated with rabbit polyclonal antibodies to collagen I (ab96723) and III (ab7778) (both from Abcam, Cambridge, MA, USA), p-Smad2 (3108), Smad2 (3122), p-Smad3 (9520), Smad3 (9523) (all from Cell Signaling Technology, Inc.), HIF-1α (ab51608; Abcam) and histone H3 (sc-8654-R; Santa Cruz Biotechnology) or mouse polyclonal α-SMA antibody (BM0002; Wuhan Boster Biological Technology, Ltd., Wuhan, China) and β-actin antibody (4970; Cell Signaling Technology, Inc.). Horseradish peroxidase-conjugated goat anti-rabbit (BA1054) or anti-mouse (BA1050) antibody (Wuhan Boster Biological Technology, Ltd.) was used as a secondary antibody. Proteins were visualized by enhanced chemiluminescence system using FluorChem FC system (Alpha Innotech, San Leandro, CA, USA).

Statistical analyses were performed using SPSS 13.0 software (SPSS, Inc., Chicago, IL, USA). Data are presented as the means ± standard error of 3 independent experiments. Statistical analysis was performed using the Student's t-test. A value of P<0.05 was considered to indicate a statistically significant difference.

We first determined the expression of HIF-1α in dermal normal and keloid tissue. As is known, HIF-1α functions as a key transcription factor in response to hypoxia (15). The results from western blot analysis (Fig. 1A) and RT-qPCR (Fig. 1B) demonstrated that the keloid tissue expressed higher levels of HIF-1α compared with normal tissue, which indicates that keloids are a relatively hypoxic tissue and that HIF signaling may play a role during the formation of keloids.

Human adult dermal fibroblasts were cultured in 21, 10, 5 or 1% oxygen for 48 h. Culturing cells in 1% oxygen significantly increased the expression of HIF-1α and stabilized nuclear HIF-1α (Fig. 2). Moreover, under 1% oxygen conditions, most of the cells that were negative in staining (Q3 area) were normal. Cells in the early apoptotic phase were stained with Annexin V but no PI and are shown in the Q4 area. Cells in the late apoptotic phase were stained with Annexin V and PI, and are shown in the Q2 area, and dead cells were stained with PI and are shown in the Q1 area. The percentage of early apoptotic cells was 0.9±0.2% in the untreated group and 0.6±0.3% in the group treated with 1% oxygen. No significant differences were observed between the cells. The percentage of late apoptotic cells was 0.2±0.06% in the control group and 0.1±0.05% in the 1% oxygen-treated group. There was also no significant differences between the control group and the 1% oxygen-treated group. There was a slight trend toward more dead cells with 1% oxygen treatment, but this did not reach statistical significance (Fig. 3). The percentage of dead cells was 1.7±0.4% in the untreated group and 2.2±0.5% in the group treated with 1% oxygen (P>0.05).

We then cultured the dermal fibroblasts with 1% oxygen alone or, 10 ng/ml TGF-β1 alone or a combination of 1% oxygen and 10 ng/ml TGF-β1 for 48 h. In addition, the levels of myofibroblast makers, α-SMA and collagen I and III, were measured by western blot analysis. As shown in Fig. 4, a significant increase in both α-SMA, collagen I and III protein expression were detected at 2 days post-treatment iwth 1% oxygen compared with the controls (P<0.05). Of note, the pro-fibrotic effects of treatment with TGF-β1 were enhanced by hypoxia. Treatment of the dermal fibroblasts with TGF-β1 significantly increased the expression of α-SMA and collagen I and III (P<0.05), and this was further enhanced when the cells were exposed to hypoxia (P<0.05).

It is well known that Smad3 phosphorylation is linked to the fibrotic process (16,17). Thus, in this study, we addressed the question of whether Smad3 activation participates in the hypoxia-induced transition of dermal fibroblasts to a myofibroblast-like phenotype. First, we cultured dermal fibroblasts with 1% oxygen alone or, 10 ng/ml TGF-β1 alone, or a combination of 1% oxygen and 10 ng/ml TGF-β1 for 48 h. In addition, the phosphorylation of Smad2 and Smad3 was measured by western blot analysis (Fig. 5) and immunofluorescence staining (Fig. 6). Our results revealed that the dermal fibroblasts exposed to 1% oxygen alone or 10 ng/ml TGF-β1 alone exhibited an increase in the activity of p-Smad2 and p-Smad3. Moreover, the levels of Smad2 and Smad3 phosphorylation were further significantly enhanced when the cells were treated when a combination of TGF-β1 and 1% hypoxia (P<0.01). Immunofluorescence staining indicated that following treatment with 1% oxygen or TGF-β1 stimulation, the complex of Smad2 and Smad3 was imported from the cytoplasm to the nucleus. Furthermore, the imported complex of Smad2 and Smad3 in the nucleus was further enhanced when the cells were treated with a combination of TGF-β1 and 1% oxygen.

To further address the question of whether the Smad3 signaling pathway is required in the hypoxia-induced transition of dermal fibroblasts to a myofibroblast-like phenotype, we used the specific inhibitor of Smad3, SIS3 (18). To demonstrate that SIS3 is effective in inhibiting the activation of Smad3 through translocation to the nucleus, protein extracts from nuclear fractions was obtained from dermal fibroblasts exposed TGF-β1 with or without SIS3. The results revealed that in TGF-β1-treated dermal fibroblasts, Smad3 expression was significantly enhanced in the nucleus, suggesting nuclear translocation. However, in the TGF-β1-treated dermal fibroblasts also treated with SIS3, the expression of Smad3 was significantly impaired, indicating that SIS3 inhibited the Smad3 nuclear translocation (Fig. 7). We then examined the effects of SIS3 on the levels of fibrotic markers induced by hypoxia. SIS3 incubation inhibited the increase in the protein level of the fibrotic markers, α-SMA and collagen I and III (Fig. 8). These results suggest that the hypoxia-induced transition of dermal fibroblasts into myofibroblasts is dependent on the TGF-β1/Smad3 pathway.