Asiaticoside Induces Human Collagen I Synthesis through TGFβ Receptor I Kinase (TβRI Kinase)-Independent Smad Signaling

• Abstract
• Abbreviations
• Introduction
• Materials and Methods
• Reagents
• Cell culture
• Quantitative detection of type I collagen
• Immunoprecipitation of Smad proteins
• Western blot analysis
• Isolation of nuclei
• Statistics
• Results
• Discussion
• Acknowledgements
• References

Abstract

Skin aging appears to be principally related to a decrease in the levels of type I collagen, the primary component of the skin dermis. Asiaticoside, a saponin component isolated from Centella asiatica, has been shown to induce type I collagen synthesis in human dermal fibroblast cells. However, the mechanism underlying asiaticoside-induced type I collagen synthesis, especially at a molecular level, remains only partially understood. In this study, we have attempted to characterize the action mechanism of asiaticoside in type I collagen synthesis. Asiaticoside was determined to induce the phosphorylation of both Smad 2 and Smad 3. In addition, we detected the asiaticoside-induced binding of Smad 3 and Smad 4. In a consistent result, the nuclear translocation of the Smad 3 and Smad 4 complex was induced via treatment with asiaticoside, pointing to the involvement of asiaticoside in Smad signaling. In addition, SB431542, an inhibitor of the TGFβ receptor I (TβRI) kinase, which is known to be an activator of the Smad pathway, was not found to inhibit both Smad 2 phosphorylation and Type 1 collagen synthesis induced by asiaticoside. Therefore, our results show that asiaticoside can induce type I collagen synthesis via the activation of the TβRI kinase-independent Smad pathway.

Abbreviations

TGF:transforming growth factor

Smad:sma- and Mad-related protein

TβRI kinase:TGFβ receptor I kinase

Introduction

Aging of the skin is primarily related to reductions in the levels of type I collagen, which is the principal component of skin dermis. Type I collagen is the main structural component of the extracellular matrix (ECM), which is known to perform a pivotal function in the maintenance of the structure of the skin dermis. Several molecules have been reported to augment type I collagen synthesis, namely, transforming growth factor-β (TGF-β) and sphingosine 1-phosphate [1], [2]. Recently, asiaticoside (Fig. [1]), which can be isolated from Centella asiatica, has been reported to augment the expression of the type I collagen gene [3].

Both the quantity and quality of extracellular collagen are determined by the balance existing between degradation and synthesis [4]. Degradation appears to be mediated by matrix metalloproteinases (MMPs) as well as by endogenous tissue inhibitors (TIMPs). Collagen synthesis is both transcriptionally and post-translationally regulated. Several studies have reported that the Smad pathway performs a function in the activation of type I collagen gene expression. The Smads are a series of proteins which perform downstream functions from the serine/threonine kinase receptors of the TGF-β family, thereby transducing signals to the nucleus. Following the binding of TGF-β to its receptors, the receptor-regulated Smads (R-Smads), Smad2, and Smad3, are phosphorylated by the type I receptor, and are known to associate with the common partner, Smad4. The resulting heteromultimer then translocates to the nucleus, in which it functions as a regulator of type I collagen gene expression [5], [6], [7].

Asiaticoside is a saponin component that has been isolated from Centella asiatica, a plant that has been used for hundreds of years in the traditional medicine of many Asiatic countries, usually for the improvement of wound healing. Recently, several studies have reported that asiaticoside enhances the synthesis of collagen [8], [9]. However, the mechanism by which asiaticoside promotes collagen synthesis, particularly at the molecular level, remains somewhat unclear.

In this report, we have demonstrated that treatment with asiaticoside induces the synthesis of type I collagen, and the mechanisms underlying its action may be mediated via the TGFβ receptor I kinase (TβRI kinase)-independent Smad activation pathway in cultured human dermal fibroblast cells.

Materials and Methods

Reagents

Asiaticoside (98 %) was purchased from LKT Laboratories, Inc. (St. Paul, MN). Tumor necrosis factor (TNF-α) was acquired from Sigma (St. Louis, MO). Anti-Phospho-Smad2 (Ser465/467) antibody was obtained from Cell Signaling Technology, Inc. (Beverly, MA). Smad2 (S-20), Smad3 (FL-425), and Smad4 (B-8) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The rabbit anti-phosphoserine antibody was acquired from Zymed Laboratories, Inc. (South San Francisco, CA)

Cell culture

Human dermal fibroblast cells (derived from neonatal foreskin) were acquired from the Amore-Pacific Corporation R&D Center, which is located in Korea. The cells were then cultured in Dulbecco’s modified Eagle's medium (Gibco, MD) containing 10 % fetal bovine serum (GibCo, MD), and penicillin-streptomycin at 37 °C, in a humidified atmosphere containing 95 % air/5 % CO₂.

Quantitative detection of type I collagen

The quantity of type I collagen in the cells was determined using a commercially available kit (Takara Bio Inc., Japan). This kit is capable of detecting procollagen type I carboxy-terminal peptide (PIP) using polyclonal antibodies, rather than directly measuring collagen. Human dermal fibroblast cells were then incubated in either the presence or the absence of asiaticoside or TGF-β, along with the indicated concentrations of SB431542 for 24 h, and then the culture supernatants were harvested and measured with a sandwich immunoassay kit, which was utilized in accordance with manufacturer’s instructions (Takara Bio Inc., Japan). The measurement was performed with a microplate reader at 450 nm.

Immunoprecipitation of Smad proteins

Smad immunoprecipitation and blotting were conducted as described [10]. In brief, the fibroblast cells were seeded in 6-well plates and then cultured for 24 h. The medium was then replaced with HEPES buffer (1 M) for 2 h. The cells were then treated with TGF-β or asiaticoside at different time periods. The fibroblast cells were rinsed twice in ice-cold PBS, and were harvested in RIPA buffer (50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1 % Nonidet P-40, 0.5 % deoxycholic acid, 0.1 % SDS), containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, 1 μg/mL leupeptin, 1 μg/mL aprotinin, and 1 μg/mL pepstatin) and phosphatase inhibitors (1 mM sodium orthovanadate, 50 mM NaF, and 40 mM β-glycerophosphate). The lysates were centrifuged at 14,000 × g for 30 min. 100 μg of lysate proteins were immunoprecipitated overnight at 4 °C with 0.2 μg of either anti-Smad2 or anti-Smad3 antibodies, followed by precipitation using 10 μL of protein G plus agarose at 4 °C for 90 min. After four washings with complete RIPA buffer, the immunoprecipitates were eluted via 5 min of boiling in 60 μL of SDS sample buffer (100 mM Tris/HCl, pH6.8, 4 % SDS, 0.2 % bromophenol blue, 20 % glycerol, 200 mM dithiothreitol).

Western blot analysis

In our Western blot analysis, the immunoprecipitates and cell lysates were separated via SDS-PAGE. The gels were blotted overnight on polyvinylidene difluoride membranes and then exposed to the appropriate antibodies. Proteins were visualized with the ECL system from Amersham Biosciences using horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibody.

Isolation of nuclei

The nuclei were isolated as was previously described [11]. In brief, the fibroblast cells were washed with PBS, scraped into PBS, then centrifuged at 5,000 × g for 5 min. The cells were then resuspended in 400 μL of buffer A (10 mM HEPES, pH 5.5, 5 mM MgCl₂, 15 mM KCl, 1 mM phenylmethylsulfonyl fluoride) at 5 × 10⁶ cells/mL, frozen in liquid nitrogen, thawed rapidly at a temperature of 37 °C, and then passed 15 times through a 25-gauge needle. The homogenate was then layered on top of 200 μL of a sucrose gradient (50 % sucrose in buffer A), and centrifuged for 3 min at 15,000 × g. Intact nuclei, which were pelleted through the cushion, were then suspended in RIPA buffer and lysed via freeze-thawing.

Statistics

The statistical significance of the data was determined by a Student’s t-test. A p < 0.01 was considered to be significant.

Results

Asiaticoside, one of the primary triterpenic compounds isolated from Centella asiatica, possesses a chemical structure containing five six-carbon rings (Fig. [1]). Several reports have shown that asiaticoside induces the synthesis of type I collagen [8], [9]. In order to verify this suggestion, we conducted a type I procollagen synthesis assay. Our type I procollagen test revealed that asiaticoside induces a significant type I collagen synthesis (Fig. [2]). As a positive control, TGF-β (10 ng/mL) was employed.

The Smad signaling cascade is known to perform an important function in human collagen production events associated with TGF-β or sphingosine 1-phosphate (S1P) [1], [2]. Therefore, as an initial step toward the elucidation of asiaticoside’s action mechanism on type I collagen synthesis, we examined its effects on phosphorylations of Smad2 and Smad3. As is shown in Fig. [3], Smad2 and Smad3 phosphorylation could be induced by treatment with asiaticoside (10 μM). Human dermal fibroblast cells were determined to respond to asiaticoside after 10 min, and this response persisted for a total of 45 min (Figs. [3] A and B). In addition, asiaticoside induced Smad2 and Smad3 phosphorylation in a concentration-dependent manner (Figs. [3] C and D). TGF-β was employed as a positive control.

In order to further substantiate asiaticoside’s particular role, we also assessed the downstream events which occurred after Smad2/3 phosphorylation. The immunoprecipitation of cell lysates with anti-Smad3 antibodies, which was followed by Western blotting for Smad3 or Smad4, revealed that levels of Smad3 expression were not influenced by TGF-β or asiaticoside treatment (data not shown). As expected, no Smad4 was shown to have been co-immunoprecipitated under control conditions. However, Smad3-Smad4 complexes were detected after stimulation with TGF-β, and also after exposure to asiaticoside (Fig. [4]).

In order to confirm the nuclear translocation of the Smad3-Smad4 complex, as was reported for TGF-β, we performed Western blotting using nuclear extracts. In this experiment, the measurement of marker enzymes localized specifically in the cytosol (lactate dehydrogenase) or the endoplasmic reticulum (a-glucosidase II) allowed us to confirm the purity of these nuclei. Western blotting of Smad3 and Smad4 in the lysates of nuclei have revealed the absence of both Smad proteins under control conditions. However, in cases in which cells were stimulated for 30 min either by TGF-β or asiaticoside, both Smad3 and Smad4 were seen to appear in the nuclear fraction (Fig. [5]), thereby indicating their translocation from the cytosol.

As has been previously mentioned, although asiaticoside induced Smad2/3 phosphorylation, the detailed mechanism underlying asiaticoside-induced Smad2/3 phosphorylation remained somewhat unclear. Therefore, as an initial step toward this end, we have attempted to characterize the relationship which exists between asiaticoside and TGF-β signaling, using SB431542, an inhibitor of the TGF-β type I receptor (TβRI), which is responsible for the phosphorylation of Smad2/3. Using this, we have examined the effects of SB431542 on the asiaticoside-induced phosphorylation of Smad2 and type I collagen synthesis. As was shown in Figs. [6] and [7], whereas SB431542 was found to block both the phosphorylation of Smad2 and type I collagen synthesis via TGF-β, the asiaticoside-induced phosphorylaton of Smad2 and type I collagen synthesis were not reduced as the result of an administration of SB431542.

Discussion

To the best of our knowledge, this study is the first to attempt to elucidate the detailed action mechanisms underlying asiaticoside-induced type I collagen synthesis. We have discovered that asiaticoside induces the synthesis of type I collagen via Smad signaling, in a TGF-β-independent manner.

Asiaticoside, one of the primary compounds contained in Centella asiatica, has been reported recently to increase the formation of extracellular matrix synthesis, including those associated with human 1(I) and 3(III) collagens [9]. In addition, we also demonstrated that asiaticoside induces a significant type I collagen synthesis in human dermal fibroblast cells (Fig. [2]). However, the precise mechanisms underlying asiaticoside-induced collagen synthesis remain somewhat unclear at the molecular level. Therefore, we have attempted to elucidate the molecular changes induced by asiaticoside in human dermal fibroblast cells. As an initial step, we have attempted to determine whether or not asiaticoside can induce both Smad2 and Smad3 phosphorylation, an initial molecular event of Smad signaling, as it is well known that Smad signaling is involved with the collagen synthesis induced by either TGF-β or sphingosine 1-phosphate. In this study, we determined that Smad2 and Smad3 were both phosphorylated by treatment with asiaticoside. In addition, after asiaticoside treatment, we observed interactions between Smad3 and Smad4, indicating the possibility that asiaticoside is involved in Smad signaling, and that it operates upstream of Smad2 and Smad3. This is further strengthened by the fact that the Smad3-Smad4 complex was translocated into the nucleus, in response to the administration of asiaticoside.

As was previously mentioned, we successfully demonstrated that asiaticoside operates upstream of Smad2 and 3. However, the relationship between asiaticoside and the signaling molecules upstream of Smad2/3 must be established in further detail. It is well known that TGFβ receptor I kinase (TβRI kinase) can phosphorylate Smad2/3 in the process of TGF-β signaling. We also attempted to determine whether or not asiaticoside-induced Smad signaling is mediated in a TβRI-dependent manner. To that purpose, SB431542, a TβRI kinase inhibitor, was introduced during an asiaticoside-induced Smad2 phosphorylation and type I collagen synthesis events. As is shown in Figs. [6] and [7], SB431542 was not determined to inhibit asiaticoside-induced Smad2 phosphorylation and type I collagen synthesis. This indicates that asiaticoside does, indeed, induce type I collagen synthesis through the activation of Smad signaling in a TβRI kinase-independent manner. Recently, signals derived from growth factor receptors which exhibited tyrosine kinase activity were also determined to modulate Smad-dependent effects. This may occur as the result of the activation of a kinase located downstream of MEK-1, and upstream of the MAPK/ERK kinase pathway, resulting in the phosphorylation of Smad2 [12]. In addition, a host of other kinases have been implicated in Smad signaling, including TAK-1 and TAB, although their precise functions have yet to be elucidated [13], [14], [15]. When taken together, these data support the conclusion that asiaticoside-induced Smad signaling is mediated by other kinases, which play the same role as TβRI kinase.

In conclusion, the data acquired in this study demonstrate that asiaticoside can induce the synthesis of type I collagen, and the mechanisms underlying its action may be mediated via the TβRI kinase-independent Smad activation pathway.

Acknowledgements

This work was supported by a grant from the Korean Ministry of Commerce, Industry, and Energy (IH-9-12-10 018 068).

References

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Prof. Yeong Shik Kim

Natural Products Research Institute

College of Pharmacy

Seoul National University

28 Yeonkun Dong

Jongro Gu

Seoul 110-460

Korea

Phone: +82-2-740-8929

Fax: +82-2-4563979

Email: kims@plaza.snu.ac.kr

References

• 1 Cuiyan X, Shuyu R, Burkhardt K, Soheyla S, Wolfgang E, Heinfried R. et al . Sphingosine 1-phosphate cross-activates the Smad signaling cascade and mimics transforming growth factor-β-induced cell responses.  J Biol Chem. 2004;  279 35 255-62
  PubMedSearch in Google Scholar
• 2 Markus B, Gero V G, Dan L, Alfredo D R, Amer A B, Marcos R. et al . A mechanism of suppression of TGF-β/SMAD signaling by NF-κB/RelA.  Genes Dev. 2000;  14 187-97
  PubMedSearch in Google Scholar
• 3 Lu L, Ying K, Wei S, Liu Y, Lin H, Mao Y. Dermal fibroblast-associated gene induction by asiaticoside shown in vitro by DNA microarray analysis.  Br J Dermatol. 2004;  151 571-8
  CrossrefPubMedSearch in Google Scholar
• 4 Dhalla A K, Hill M F, Singal P K. Role of oxidative stress in transition of hypertrophy to heart failure.  J Am Coll Cardiol. 1996;  28 506-14
  PubMedSearch in Google Scholar
• 5 Piek E, Heldin C H, Ten Dijke P. Specificity, diversity, and regulation in TGF-ß superfamily signaling.  FASEB J. 1999;  13 2105-24
  PubMedSearch in Google Scholar
• 6 Attisano L, Wrana J L. Smads as transcriptional co-modulators.  Curr Opin Cell Biol. 2000;  12 235-43
  CrossrefPubMedSearch in Google Scholar
• 7 Massagué J, Wotton D. Transcriptional control by the TGF-ß/Smad signaling system.  EMBO J. 2000;  19 1745-54
  CrossrefPubMedSearch in Google Scholar
• 8 Bonte F, Dumas M, Chaudagne C, Meybeck A. Influence of asiatic acid, madecassic acid, and asiaticoside on human collagen I synthesis.  Planta Med. 1994;  60 133-5
  PubMedSearch in Google Scholar
• 9 Lu L, Ying K, Wei S, Fang Y, Liu Y, Lin H. et al . Asiaticoside induction for cell-cycle progression, proliferation and collagen synthesis in human dermal fibroblasts.  Int J Dermatol. 2004;  43 801-7
  CrossrefPubMedSearch in Google Scholar
• 10 Poncelet A C, de Caestecker M P, Schnaper H W. The transforming growth factor-beta/SMAD signaling pathway is present and functional in human mesangial cells.  Kidney Int. 1999;  56 1354-65
  CrossrefPubMedSearch in Google Scholar
• 11 Bunce C M, Thick J A, Lord J M, Mills D, Brown G. A rapid procedure for isolating hemopoietic cell nuclei.  Anal Biochem. 1988;  175 67-73
  CrossrefPubMedSearch in Google Scholar
• 12 Brown J D, DiChiara M R, Anderson K R, Gimbrone MA J r, Topper J N. MEKK-1, a component of the stress (stress-activated protein kinase/c-Jun N-terminal kinase) pathway, can selectively activate Smad2-mediated transcriptional activation in endothelial cells.  J Biol Chem. 1999;  274 8797-805
  CrossrefPubMedSearch in Google Scholar
• 13 Shibuya H, Yamaguchi K, Shirakabe K, Tonegawa A, Gotoh Y, Ueno N. et al . An activator of the TAK1 MAPKKK in TGF-β signal transduction.  Science. 1996;  272 1179-82
  PubMedSearch in Google Scholar
• 14 Shirakabe K, Yamaguchi K, Shibuya H, Irie K, Matsuda S, Moriguchi T. et al . TAK1 mediates the ceramide signaling to stress-activated protein kinase/c-Jun N-terminal kinase.  J Biol Chem. 1997;  272 8141-4
  CrossrefPubMedSearch in Google Scholar
• 15 Yamaguchi K, Shirakabe K, Shibuya H, Irie K, Oishi I, Ueno N. et al . Identification of a member of the MAPKKK family as a potential mediator of TGF-β signal transduction.  Science. 1995;  270 2008-11
  PubMedSearch in Google Scholar

Prof. Yeong Shik Kim

Natural Products Research Institute

College of Pharmacy

Seoul National University

28 Yeonkun Dong

Jongro Gu

Seoul 110-460

Korea

Phone: +82-2-740-8929

Fax: +82-2-4563979

Email: kims@plaza.snu.ac.kr