As previously described (14,15), wild-type mice (C57BL/6J, male, 8 weeks old; n=32) were purchased from Vital River Laboratory Animal Technology (Beijing, China) and housed in a pathogen-free and light-controlled room (12 h light and 12 h dark) with free access to food and water. BLM sulfate from Hospira, Inc. (Lake Forest, IL, USA) was used to induce fibrosis. The mice were anesthetized (with a 3 ml/kg mixture of 25 mg/kg of ketamine in 2.5 ml of xylazine), followed by treatment with either saline (n=3–4 mice) or BLM (n=3–4 mice) (1.5 U/kg of body weight) in saline solution through an intratracheal (IT) injection. Twenty-one days post-BLM administration, the animals were sacrificed (by cervical dislocation following CO₂ inhalation), and the lungs were removed for histological staining and the isolation of lung fibroblasts. All animal experiments were performed in accordance with protocols approved by the Animal Care and Use Committee of Xuzhou Medical College, Xuzhou, China.

The lung tissues were fixed in formalin and then embedded in paraffin. Thereafter the samples were cut into 5-µm-thick sections. After removing the paraffin, the mouse lung tissue sections (5-µM-thick) were hydrated and washed in deionized water followed by block solution [Tris-buffered saline (TBS) with 1% fetal bovine serum (FBS) 1 h] at room temperature, incubated with goat anti-HSP27 antibody and rabbit anti-fibronectin (FN) antibody (1:500 in the block solution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at 4°C overnight, and incubated with Alexa Fluor secondary antibodies (1:200 dilutions in blocking buffer) for 1 h, followed by mounting; and examined under a Nikon Eclipse TE 2000-S fluorescence microscope with a 60X oil immersion objective lens. For human lung fibroblasts, the treated cells were fixed and permeabilized, then incubated with primary antibodies (1:200 dilutions in blocking buffer) for 1 h and washed 3 times, thereafter incubated with Alexa Fluor secondary antibodies (Alexa Fluor 594 for HSP27 and Alexa Fluor 488 for Fibronectin; Biolegend, San Diego, CA, USA; 1:200 dilutions in blocking buffer) for another 1 h. The nuclei were counterstained with DAPI (0.5 µg/ml; Sigma-Aldrich, Inc., St. Louis, MO, USA). After washing, the slides were mounted on slides and examined under a fluorescence microscope (Olympus IX83; Olympus, Tokyo, Japan), as previously described (16,17).

Primary mouse lung fibroblasts were isolated from the C57BL/6J mice treated with or without BLM as previously described (16), and the isolated cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS for 14 days. The WI-38 cells (a human lung fibroblast cell line) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and the cells were grown and maintained in 6-well plates with DMEM medium containing 10% FBS.

Recombinant human TGF-β1 was obtained from Peprotech, Inc. (Rocky Hill, NJ, USA) and dissolved in phosphate-buffered saline (PBS) solution. Serum-starved lung fibroblasts (~80% confluent) were treated with TGF-β (5 ng/ml) for 0–48 h. NF-κB inhibitor was used as previously described (16). In detail, NF-κB inhibitor (Bay 11-7082), which was obtained from Sigma-Aldrich, Inc., was applied to the lung fibroblasts at a final concentration of 10 µM for 1 h, as previously described (16). The NF-κB inhibitor was dissolved in DMSO; DMSO was used as a parallel control to eliminate the influence of DMSO itself. The cells were subsequently challenged with TGF-β (5 ng/ml) for 48 h, and cell lysates (20 µg protein) were subjected to western blot analysis.

The scrambled RNA (scRNA), siRNA targeting human NF-κB p65 subunit (si-p65) and siRNA targeting the human Smad3 (si-Smad3) smart pool, used in this study were from Santa Cruz Biotechnology, Inc. Vehicle shRNA (Veh shRNA) and shRNA targeting human HSP27 shRNA (HSP27 shRNA) were from Sigma-Aldrich, Inc. For target gene depletion, the WI-38 cells cultured on 6-well plates (50–60% confluence) were transiently transfected with 200 nmol/l of siRNA (scRNA, si-p65 or si-Smad3), or transfected with 3 µg/well of shRNA (Veh shRNA or HSP27 shRNA) using Attractene transfection reagent according to the manufacturer's instructions (Qiagen, Inc., Gaithersburg, MD, USA). After 48 h, the cells were further treated with TGF-β (5 ng/ml) for a further 0–48 h. Protein expression was analyzed by western blot analysis or under a fluorescence microscope, and the mRNA levels were analyzed by reverse transcription-quantitative PCR (RT-qPCR).

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis were performed as previously described (16,18). The protease inhibitor cocktail tablets (EDTA-free complete) were from Roche Diagnostics (Indianapolis, IN, USA). Mouse anti-α-smooth muscle actin (α-SMA) and anti-β-actin antibodies were from Sigma-Aldrich, Inc. Cell lysis buffer and rabbit anti-Smad3 antibody was from Cell Signaling Technology, Inc. (Danvers, MA, USA). Rabbit anti-FN and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies, mouse anti-p65 antibody and goat anti-HSP27 antibody were purchased from Santa Cruz Biotechnology, Inc. Horseradish peroxidase (HRP)-linked anti-mouse IgG and anti-rabbit IgG antibodies were obtained from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). Briefly, cell lysates were prepared in lysis buffer containing EDTA-free complete protease inhibitors, followed by centrifugation at 10,000 × g for 10 min, boiled with Laemmli sample buffer for 5 min, separated on 10 or 4–20% SDS-PAGE, transferred onto PVDF membranes, and blocked with TBST containing 5% BSA prior to incubation with primary antibodies (1:1,000 dilution) overnight, and secondary antibodies (1:2,000 dilutions) for 2 h at room temperature. The blots were developed and quantified using ImageQuant 5.2 software (Molecular Dynamics, Sunnyvale, CA, USA).

Total RNA was isolated from the cells using TRIzol reagent (Life Technology, Rockville, MD, USA), and RNA (1 µg) was reversed transcribed using the cDNA synthesis kit (Bio-Rad Laboratories, Inc.). Quantitative PCR (qPCR) was performed as previously described (14,16). Briefly, qPCR detection was performed using the ABI 7500 system (Life Technologies, Grand Island, NY, USA) with the SYBR-Green supermix kit (Bio-Rad Laboratories, Inc.). According to the manufacturer's instructions, the running parameters were set as denaturation at 95°C for 3 min, 40 cycles of denaturation at 95°C for 15 sec and annealing/extension at 60°C for 1 min, finally the melt curve analysis was set as 95°C for 15 sec, 60°C for 1 min and 95°C for 15 sec. For human Smad3, the primer sequence was forward, 5′-TGGACGCAGGTTCTCCAAAC-3′ and reverse, 5′-CCGGCTCGCAGTAGGTAAC-3′; for human GAPDH, the primer sequence was forward, 5′-TGTGGGCATCAATGGATTTGG-3′ and reverse, 5′-ACACCATGTATTCCGGGTCAAT-3′. GAPDH was used as a housekeeping gene to normalize the expression levels as previously described (14).

Primary data from at least 3 independent experiments are expressed as the means ± SEM, and subjected to statistical analysis using the two-tailed Student's t-test or one-way ANOVA. Values of P<0.05 were considered to indicate statistically significant differences (19,20).

The role of HSP27 in the etiology of lung fibrosis is elusive. In this study, we examined the expression of HSP27 in lung tissue from C57BL/6J mice challenged with BLM by IT injection (male, 8 weeks old) or treated with saline. At 21 days post-challenge, lung tissues were isolated from the mice and and used for immunofluorescence staining for HSP27 and FN as described in the Materials and methods. As shown in Fig. 1A, the expression of HSP27 was significantly increased and co-localized with FN, a marker of fibrogenesis, in the lung fibrotic foci from the BLM-challenged mice. The resutls of western blot analysis also indicated that HSP27 expression was markedly increased in the lung tissue from the BLM-challenged mice (Fig. 1B and C).

We then examined the expression of HSP27 in lung fibroblasts from the control and BLM-challenged mice. As shown in Fig. 2, the expression levels of HSP27, as well as that of bio-markers of fibroblast differentiation (α-SMA and FN), were markedly higher in the lung fibroblasts from BLM-challenged mice than in those from the control mice. These findings suggest that HSP27 expression in lung fibroblasts may be associated with fibroblast differentiation under fibrotic conditions.

TGF-β is a key factor for fibroblast differentiation through the regulation of the expression of various genes during pulmonary fibrosis (21). Thus, we then investigated whether HSP27 expression is regulated by TGF-β in lung fibroblasts. As shown in Fig. 3A and B, the TGF-β challenge (5 ng/ml, 48 h) markedly increased HSP27 expression and promoted fibroblast differentiation. Furthermore, we confirmed the expression of HSP27 by immunofluorescence staining. Examination under a fluorescence microscope also indicated that the TGF-β challenge upregulated the expression of HSP27 and FN in human lung fibroblasts (Fig. 3C).

To further determine the molecular mechanisms through which TGF-β regulates HSP27 expression in lung fibroblasts, we knocked down the expression of Smad3 or the NF-κB p65 subunit, which are the key transcription factors in TGF-β-challenged lung fibroblasts. Transfection with si-Smad3 significantly decreased the mRNA (Fig. 4A) and protein expression (Fig. 4B and C) of Smad3 in the human lung fibroblasts, and also inhibited the TGF-β-induced HSP27 expression in the human lung fibroblasts (Fig. 4B and C). Similarly, the knockdown of the p65 subunit by transfection with si-p65 also markedly inhibited the TGF-β-induced HSP27 expression and the differentiation of human lung fibroblasts (Fig. 5A and B). Additionally, treatment with NF-κB inhibited the TGF-β-induced increase in HSP27 expression and the differentiation of human lung fibroblasts (Fig. 5C and D). Taken together, these data indicated that TGF-β induced HSP27 expression through the Smad3 and NF-κB pathways in human lung fibroblasts.

We also examined the role of HSP27 in TGF-β-induced fibroblast differentiation. As shown in Fig. 6, transfection with HSP27 shRNA significantly decreased the expression of HSP27 in human lung fibroblasts, and also inhibited the TGF-β-induced differentiation of lung fibroblasts (Fig. 6). The TGF-β-induced activation of Smad3 and ERK are critical for lung fibroblast differentiation during pulmonary fibrogenesis (6,14). We then further examined whether HSP27 regulates the TGF-β-induced activation of the Smad and ERK signaling pathways in human lung fibroblasts. As shown in Fig. 7, the knockdown of HSP27 markedly blocked the TGF-β-induced activation of Smad3 and ERK. Additionally, the role of HSP27 in the TGF-β-induced activation of Akt, p38 and JNK was examined studied. However, the knockdown of HSP27 had shown no effect on the TGF-β-induced activation of Akt, p38 and JNK (data not shown). Taken together, these data indicate that HSP27 regulates TGF-β-induced human lung fibroblast differentiation through the regulation of Smad3 and ERK activation.