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β-Lapachone Regulates the Transforming Growth Factor-β–Smad Signaling Pathway Associated with Collagen Biosynthesis in Human Dermal Fibroblasts
2016 年 39 巻 4 号 p. 524-531
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2016 年 39 巻 4 号 p. 524-531
The transforming growth factor (TGF)-β–Smad signaling pathway regulates collagen biosynthesis in human dermal fibroblasts. We found that β-lapachone stimulated type I collagen expression in human dermal fibroblasts. In this study, we evaluated whether the β-lapachone-induced upregulation of collagen biosynthesis in human dermal fibroblasts is associated with the TGF-β–Smad signaling pathway. In cultured human dermal fibroblasts, both Smad 2 and Smad 3 (Smad 2/3) were phosphorylated by β-lapachone treatment in a concentration-dependent manner. SB431542, a specific inhibitor of TGF-β receptor I kinase, inhibited the β-lapachone-mediated Smad 2/3 phosphorylation and type I collagen expression, suggesting that β-lapachone stimulates collagen production via the TGF-β receptor I kinase-dependent pathway. β-Lapachone did not increase TGF-β1 synthesis in human dermal fibroblasts, suggesting that the molecular mechanism of β-lapachone for the upregulation of collagen synthesis is due to the extracellular regulation of availability and activities of TGF-β. This study provides new insights into the role of β-lapachone in collagen synthesis in human dermal fibroblasts and suggests that β-lapachone can be used as a pharmacological tool to study collagen homeostasis associated with TGF-β–Smad signaling.
Skin regeneration treatments are of considerable interest to cosmeceutical researchers and dermatologists for treating skin damage that is primarily induced by aging and exposure to sunlight. Various noninvasive treatments and topical cosmeceuticals are used to treat symptoms of photo-aged skin, including wrinkles.1–3) Aging can be induced by intrinsic and extrinsic factors that reduce collagen synthesis and increase the levels of matrix metalloproteinases, which results in degradation of the connective tissue network.4–6)
Type I collagen is the most abundant structural protein in skin connective tissue and it confers strength and resilience to skin. Type I collagen is primarily synthesized by fibroblasts residing within the skin connective tissue. Type I procollagen, a soluble precursor of collagen, is secreted by fibroblasts and then proteolytically processed to form insoluble collagen fibers. Disorganization, fragmentation, and dispersion of collagen bundles are prominent features of photodamaged human skin.7,8)
Transforming growth factor (TGF)-β regulates extracellular matrix (ECM) metabolism and genesis of tissue fibrosis through overproduction of type I collagen.9–12) Several signaling pathways, including the Smad and ERK pathways, mediate TGF-β-induced extracellular matrix production and fibrosis.13–15) Members of the TGF-β family regulate numerous cellular activities, including cell differentiation and ECM production,16,17) and are the primary stimulators for ECM production. In addition, other cytokines, such as connective tissue growth factor and interleukin-4, may also contribute to elevated collagen deposition.18) In fibroblasts, TGF-β functions as a potent inducer of matrix synthesis by activating the expression of ECM genes, such as the type I collagen encoding genes.19)
TGF-β signaling is initiated by activation of two types of transmembrane serine/threonine kinase receptors, TGF receptor type I (TGFβRI) and type II (TGFβRII), which are coupled to the Smad pathways.20) After TGF-β binds TGFβRII and activates TGFβRI, the cytoplasmic Smads [R-Smads; Smad 2 and Smad 3 (Smad 2/3)] are phosphorylated. The phosphorylated R-Smads form a heteromultimer with the common mediator Smad (Smad 4), which can translocate to the nucleus and then associate with transcription factor complexes to regulate the transcription of genes such as the type I collagen gene.
β-Lapachone is an o-naphthoquinone obtained from the inner bark of the lapacho tree (Tabebuia avellanedae). Its inner bark is often used as an analgesic, anti-inflammatory, antineoplastic, antimicrobial, and diuretic substance in the northeast of Brazil.21,22) β-Lapachone inhibits several types of carcinoma cells, including those isolated from hepatomas, osteosarcomas, breast cancers, prostate cancers, and human leukemias.23,24) In this study, we investigated the fibrillogenetic effect of β-lapachone on human dermal fibroblasts (HDFs). In the previous study, β-lapachone can increase cell proliferation, including keratinocytes (mouse, human), fibroblasts (mouse), and human endothelial cells. Each of the cells showed dramatic cell proliferation except human fibroblasts.25) And, β-lapachone can stimulate wound healing and has potential for therapeutic applications.25) The synthesis of ECM is a critical feature of wound healing.26) Furthermore, type I collagen is the major structural component of ECM.27) We report here that β-lapachone promotes collagen synthesis in HDFs by activating a TGFβRI kinase-dependent Smad signaling cascade. Our data helped elucidate a new signaling mechanism for β-lapachone-induced collagen biosynthesis.
β-Lapachone was purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). The chemical structure of β-lapachone is shown in Fig. 1. TGF-β1 was obtained from Roche Diagnostics (Indianapolis, IN, U.S.A.). HDF cells were obtained from Life Technologies (Carlsbad, CA, U.S.A.). Fetal bovine serum (FBS) and antibiotic/antimycotic (AA) solution were purchased from Life Technologies. Antibodies specific for collagen type I were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, U.S.A.). Dulbecco’s modified Eagle’s medium (DMEM), phosphate-buffered saline (PBS) without calcium and magnesium, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and other chemicals were purchased from Sigma-Aldrich.
HDF cells were cultured in DMEM supplemented with 10% FBS and 1% AA solution in humidified atmosphere containing 5% CO2 in air at 37°C. The cell passage number of cells used for the study was between 10 and 13, and the culture medium was changed every 2 d.
Cell Viability AssayThe MTT assay was performed to determine cell viability. HDFs were seeded in 96-well plates at a concentration of 5×103 cells/well and cultured for 24 h. The cells were incubated with and without β-lapachone for 24 and 48 h, after which the culture medium was removed and replaced with 1 mg/mL MTT solution dissolved in DMEM and incubated for an additional 4 h. The MTT solution was then removed and dimethyl sulfoxide was added, and the absorbance of the dissolved formazan crystals was measured at 570 nm wavelength using a VersaMax ELISA microplate reader (Molecular Devices, Sunnyvale, CA, U.S.A.).
Collagen AssayHDFs were seeded in 12-well plates (1×105 cells/well) and cultured in a serum-free medium for 24 h, followed by replacement with fresh medium. The cells were then cultured with and without β-lapachone. The aliquots were assayed for procollagen type I levels by using a Procollagen Type I C-Peptide EIA Kit (TaKaRa, Shiga, Japan). The assay was performed according to the manufacturer’s protocol.
Real Time Reverse Transcription Polymerase Chain Reaction (rRT-PCR)For rRT-PCR analysis, HDFs (1×105 cells/well) were cultured with and without β-lapachone. Total cellular RNA was extracted with TRIzol reagent (Life Technologies) and reverse-transcribed using a MuLV reverse transcriptase Kit (Promega, Madison, WI, U.S.A.) using the manufacturer’s protocol. The cDNA was synthesized using 1 µg of total RNA, 200 U of reverse transcriptase (M-MLV RT), and 20 pM oligo-dT. The oligonucleotide primers used were as follows: COL1A (5′-CCC GGG TTT CAG AGA CAA CTT C-3′ and 5′-TCC ACA TGC TTT ATT CCA GCA ATC-3′) and β-actin (5′-GGA CTT CGA GCA AGA GAT GG-3′ and 5′-AGC ACT GTG TTG GCG TAC AG-3′). PCR reactions were performed in a final volume of 20 µL and the reaction mix contained 2 µL of cDNA, 15 mM MgCl2, 1.25 mM deoxynucleotide triphosphate (dNTP), 20 pM of each primer, and 0.5 U of Taq polymerase (Promega). The PCR amplification was performed using 40 cycles as follows: initial denaturation at 94°C for 5 min, followed by 40 cycles at 94°C for 30 s, 50°C for 30 s, and 72°C for 30 s. Amplification was terminated by a final extension at 72°C for 5 min. β-Actin mRNA expression was used as an internal standard for all samples (TaKaRa Bio, Otsu, Japan).
Western Blot AnalysisCells were lysed with radio immunoprecipitation assay (RIPA) buffer (50 mM Tris–HCl, 0.15 M NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1% Triton X-100, 1% sodium dodecyl sulfate (SDS), 50 mM NaF, 1 mM Na3VO4, 5 mM dithiothreitol, 1 mg⁄mL leupeptin, and 20 mg⁄mL phenylmethylsulfonyl fluoride (PMSF), pH 7.4). Electrophoretic separation of 50 µg of protein was performed using SDS-polyacrylamide gel. Proteins were transferred to polyvinylidene difluoride (PVDF) membranes and incubated with antibodies to phospho-Smad 2, total-Smad 2, phospho-Smad 3, total-Smad 3 (Cell Signaling Technology, Danvers, MA, U.S.A.), collagen type I, or β-actin (Santa Cruz Biotechnology). The membranes were then washed and incubated with a horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (IgG) antibody (Cell Signaling Technology). Blots were reacted with Immobilon Western Reagent (Millipore, Billerica, MA, U.S.A.) and exposed to photographic film.
TGF-β1 Enzyme-Linked Immunosorbent Assay (ELISA) AssayHDFs were inoculated into 12-well plates (1×105 cells/well) and cultivated in serum-free medium for 24 h. The medium was then replaced with serum-free medium with or without β-lapachone. The levels of TGF-β1 were measured at different time points using a Human TGF-beta 1 Quantikine ELISA kit (R&D Systems, Minneapolis, MN, U.S.A.). The assay was performed according to the manufacturer’s protocol.
Statistical AnalysisAll data are expressed as the mean±standard deviation (S.D.) of triplicate determination. The differences between multiple groups were evaluated by one-way or two-way ANOVA followed by Dunnett’s post-hoc test and Tukey’s post-hoc test. A p value of 0.05 or lower was considered statistically significant.
To evaluate the effects of β-lapachone on collagen synthesis, primary dermal fibroblasts were incubated with β-lapachone for 24 h, and the levels of total soluble collagen were measured using an ELISA designed to detect mammalian collagens. Treatment with β-lapachone significantly increased collagen secretion (Fig. 2A). The minimum effective concentration for the collagen synthesis might be between 0.05 and 0.075 µg/mL. Especially, 0.1 µg/mL β-lapachone induced a higher level of collagen secretion than that induced by TGF-β1 (10 ng/mL). Regardless of the β-lapachone concentration, significant cytotoxicity was not observed while incubation time might affect it (Fig. 2B).
(A) Effects of β-lapachone on collagen secretion. Serum-starved human dermal fibroblasts (HDFs) were treated with β-lapachone (0–0.1 µg/mL) or TGF-β1 (10 ng/mL) for 24 h, and collagen levels in supernatants were measured using a Procollagen Type I C-Peptide (PIP) EIA kit. Results are expressed as percentages of control. (B) Cytotoxicity of β-lapachone. HDFs were cultured in the presence of various concentrations of β-lapachone (0–0.1 µg/mL) or TGF-β1 (10 ng/mL) for 24 h and 48 h. Viability was assessed using the MTT assay. Results are expressed as percentages of the control. (C) Effects of β-lapachone on type I collagen mRNA expression in HDFs. Serum-starved HDFs were cultured in the presence of various concentrations of β-lapachone (0–0.1 µg/mL) or TGF-β1 (10 ng/mL) for 3–12 h. COL1A transcription was determined using RT-PCR. (D) Effects of β-lapachone on type I collagen protein expression in HDFs. Serum-starved HDFs were cultured in the presence of various concentrations of β-lapachone (0–0.1 µg/mL) for 24 h. Cell lysates were evaluated by performing Western blot analysis by using antibodies against type I collagen or β-actin. The densities of type I collagen and β-actin were quantified from 3 independent experiments and type I collagen expression was normalized to β-actin levels in the samples. Values represent the mean±S.D. of triplicate determinations. * p<0.05, ** p<0.01 vs. control group.
β-Lapachone treatment of HDF cells significantly increased the collagen type I mRNA levels (Fig. 2C), indicating that it induces signal transduction involved in type I collagen gene expression. These results were corroborated by performing Western blot analysis of whole cell lysates (Fig. 2D). Thus, β-lapachone acts at the transcriptional level to enhance the synthesis of type I collagen.
β-Lapachone-Stimulated Phosphorylation of Smad 2/3To further elucidate the molecular pathways involved in β-lapachone-induced type I collagen synthesis, we determined the effects of β-lapachone on Smad 2/3 phosphorylation and showed that both Smad proteins were phosphorylated in β-lapachone-treated HDFs (Fig. 3). These data indicate that Smad 2/3 may play a key role in β-lapachone-induced type I collagen synthesis.
Serum-starved HDFs were treated with β-lapachone at the indicated doses (0–0.1 µg/mL) for 5 min. TGF-β1 (10 ng/mL) served as a positive control. (A) Cell lysates were assessed by performing Western blot analysis using antibodies against Smad 2, Smad 3, phospho-Smad 2, phospho-Smad 3, and β-actin. (B) and (C) The densities of Smad 2, Smad 3, phospho-Smad 2, phospho-Smad 3, and β-actin were quantified from 3 independent experiments. The levels of phosphorylation or expression of Smad 2/3 were normalized to total β-actin levels in the samples. Values represent the mean±S.D. of triplicate determinations. ** p<0.01 vs. control group.
We assessed whether β-lapachone acts as a direct or indirect activator to induce phosphorylation of Smad 2/3 in HDF cells. TGF-β-induced activation of TGFβRI involves transient interactions between TGFβRI and R-Smads, which become phosphorylated at 2 serine residues within their C termini.28) To examine the effects of β-lapachone on TGF-β-receptor activation, we assessed whether SB431542, a specific inhibitor of TGFβRI kinase that phosphorylates Smad 2/3, inhibited β-lapachone mediated Smad 2/3 phosphorylation. SB431542-treated cells showed significantly lower levels of phosphorylation than those of TGF-β1-treated control cells (Fig. 4). Moreover, treatment of HDFs with β-lapachone in the presence of SB431542 also reduced Smad 2/3 phosphorylation at the C termini. Further, the correlation between the activity of TGFβRI kinase and the β-lapachone-induced fibrotic response, including increased expression of type I collagen was evaluated. Thus, SB431542 inhibited both β-lapachone induced and TGF-β1 induced expression of type I collagen mRNA and protein levels (Fig. 5).
Serum-starved HDFs were treated with β-lapachone at concentration of 0.1 µg/mL for 5 min in the presence of SB431542 (5 or 10 µM) or vehicle. TGF-β1 (1 ng/mL) served as a positive control. (A) Cell lysates were examined by Western blotting using antibodies against Smad 2, Smad 3, phospho-Smad 2, phospho-Smad 3, and β-actin. (B) and (C) The densities of Smad 2, Smad 3, phospho-Smad 2, phospho-Smad 3, and β-actin were quantified from 3 independent experiments, and the levels of phosphorylation or expression of Smad 2/3 were normalized to total β-actin levels of the samples. Values represent the mean±S.D. of triplicate determinations. * p<0.05, ** p<0.01.
Serum-starved HDFs were treated with β-lapachone (0.1 µg/mL) or TGF-β1 (1 ng/mL) in the presence of SB431542 (10 µM) or vehicle for 24 h. (A) Cell lysates were analyzed by Western blotting using antibodies against type I collagen, and β-actin. (B) The densities of type I collagen and β-actin were quantified from 3 independent experiments, and type I collagen expression levels were normalized to total β-actin levels in the samples. (C) COL1A transcription was determined using RT-PCR. Values represent the mean±S.D. of triplicate determinations. * p<0.05, ** p<0.01.
TGF-β is a major profibrotic cytokine that stimulates dermal fibroblast proliferation and induces type I procollagen production.29,30) Therefore, we investigated whether β-lapachone stimulated the secretion of TGF-β1. Treatment of β-lapachone for various incubation periods did not increase the secretion of TGF-β1 (Fig. 6). Overall, β-lapachone did not stimulate TGF-β1 secretion in HDF cells.
Serum-starved HDFs were treated with β-lapachone or vehicle for 2, 4, 6, 12, 24, or 48 h. The TGF-β levels in supernatants at different time points (2–48 h) were measured using an ELISA kit.
This study demonstrates that β-lapachone induces type I collagen synthesis in HDFs through direct phosphorylation of Smad 2/3 initiated by TGFβRI signaling. In preliminary experiments, we assessed whether β-lapachone induces the synthesis of total collagen in human primary dermal fibroblasts and showed that β-lapachone activated the secretion of collagenous fibers in a monolayer culture of HDFs (Fig. 2A). In human skin, collagen, particularly type I, is the most abundant protein and is fundamentally implicated in skin aging.31) In this study, we showed that β-lapachone elevated the expression of type I collagen (Figs. 2C, D). In the cytotoxicity study, no significant cell viability differences were found in various concentrations of β-lapachone. Cell viability difference was observed between two different incubation time (24, 48 h). Even though, the cell viability difference in different incubation time was considered statistically significant, it didn’t imply toxicity of β-lapachone. After the 48 h incubation, average cell viability was 98.6%, which means practically no change in cell viability during the experiment (Fig. 2B).
Collagen synthesis is regulated by transcriptional and post-translational mechanisms. The latter includes hydroxylation of proline and lysine residues in procollagen polypeptide chains.32,33) Several lines of evidence indicate that among cytokines, TGF-β is the most potent inducer of type I collagen expression.20) The Smad-signaling pathway plays a central role in the transcriptional response to TGF-β signaling, such as the activation of type I collagen gene expression.34) As mentioned previously, β-lapachone significantly elevated type I collagen gene expression (Fig. 2C). Moreover, we determined that Smad 2/3 were phosphorylated in HDFs treated with β-lapachone (Fig. 3), indicating that it elevates type I collagen synthesis through the activation of Smad signaling.
The Smad signaling pathway is crucial for the activation of genes that encode fibrillar collagen. Previous studies indicate that Smad 3 plays an important role in the TGF-β responsiveness of the type I collagen gene.34–36) We then determined whether β-lapachone-activating Smad pathways are TGFβRI kinase-dependent in HDFs. Notably, several recent studies have shown that Smad signaling can be activated by sphingosine-1-phosphate, asiaticoside, and α-lipoic acid28,37,38) indicating that Smad phosphorylation can occur via a TGFβRI kinase-independent Smad-activation pathway.28,37) Lee et al. reported that TGFβRI has dual-specific kinase activities mediated by serine/threonine kinase and tyrosine kinase.39) Therefore, serine/threonine kinase activity of TGFβRI may mediate phosphorylation of Smad 3, whereas tyrosine kinase may phosphorylate phosphatidyl inositol 3-kinase (PI3K). Our results demonstrated that β-lapachone induced Smad 3 phosphorylation via the TGF-β pathway. Thus, β-lapachone may activate serine/threonine kinase of TGFβRI. Therefore, β-lapachone may have activated Smad signaling by stimulating serine/threonine kinase activity of TGFβRI.
Notably, SB431542, a specific TGFβRI kinase inhibitor, suppressed both β-lapachone and TGF-β-induced Smad 2/3 phosphorylation in HDFs (Fig. 4). Furthermore, the data indicate that the expression of type I collagen was positively regulated by a TGFβRI kinase-dependent pathway (Fig. 5). Therefore, our results indicate that β-lapachone increases type I procollagen synthesis by activating TGF-β–Smad signaling.
To further investigate the fibrillogenetic mechanism underlying the β-lapachone-induced collagen synthesis in HDFs, we assessed whether β-lapachone stimulated the secretion of TGF-β1 in HDFs. The data indicated that β-lapachone did not induce TGF-β1 secretion (Fig. 6). It has been reported that bioactivity of TGF-β is regulated by various extracellular modulators. Some of these factors enhance TGF-β signals through regulation of ligand–receptor interaction, other factors control storage, maturation, stabilization, transport, and release of TGF-β ligands.40) Thus, β-lapachone may have affected extracellular factors which regulate availability and activities of TGF-β ligand as indicated by the increased Smad 2/3 phosphorylation without elevation in TGF-β1 secretion. Further studies are required for detailed characterization of the underlying mechanisms.
In conclusion, we showed that β-lapachone induces collagen expression by activating the TGFβRI/Smad signaling cascade through TGFβRI kinase and not through the activation of a TGF-β paracrine signaling loop. To our knowledge, this is the first study to demonstrate the fibrogenic activity of β-lapachone in HDFs. Thus, these results corroborate the previous clinical observations of skin rejuvenating effects of β-lapachone. Furthermore, β-lapachone can be used to develop potential therapeutic agents for preventing and reversing the effects of intrinsic and extrinsic skin aging.
The authors declare no conflict of interest.
