Differential Regulation by IL-1β and EGF of Expression of Three Different Hyaluronan Synthases in Oral Mucosal Epithelial Cells and Fibroblasts and Dermal Fibroblasts: Quantitative Analysis Using Real-Time RT-PCR
2004; Elsevier BV; Volume: 122; Issue: 3 Linguagem: Inglês
10.1111/j.0022-202x.2004.22332.x
ISSN1523-1747
AutoresYoichi Yamada, Naoki Itano, Ken‐ichiro Hata, Minoru Ueda, Koji Kimata,
Tópico(s)Immune Response and Inflammation
ResumoUsing "real-time RT-PCR", we assessed the expression of three different hyaluronan synthase genes, HAS1, HAS2, and HAS3, by measuring their mRNA amounts in cultured human oral mucosal epithelial (COME) cells, oral mucosal fibroblasts, and dermal fibroblasts, and investigated the effects of interleukin-1β (IL-1β) and epidermal growth factor (EGF). When COME cells were treated with IL-1β or EGF, early and marked increases and subsequent rapid decreases were observed for all HAS genes and, moreover, actual changes in hyaluronan synthesis subsequently occurred. The effects of IL-1β stimulation were concentration-dependent and the maximal response to the EGF stimulation was observed at a low concentration (0.1 ng per mL). When two different types of fibroblasts were treated with IL-1β or EGF, increased expression with different degrees and rates of three different HAS genes and subsequent increased synthesis of hyaluronan were also observed. In addition, HAS1 gene expression was not detectable in the mucosal fibroblasts, while weak HAS3 gene expression was detected in the dermal fibroblasts. Taken together, it is likely that the regulation of the expression of the three different HAS genes is different between oral mucosa and skin, which may be of significance for elucidating some of the differences between these tissues in wound healing. Using "real-time RT-PCR", we assessed the expression of three different hyaluronan synthase genes, HAS1, HAS2, and HAS3, by measuring their mRNA amounts in cultured human oral mucosal epithelial (COME) cells, oral mucosal fibroblasts, and dermal fibroblasts, and investigated the effects of interleukin-1β (IL-1β) and epidermal growth factor (EGF). When COME cells were treated with IL-1β or EGF, early and marked increases and subsequent rapid decreases were observed for all HAS genes and, moreover, actual changes in hyaluronan synthesis subsequently occurred. The effects of IL-1β stimulation were concentration-dependent and the maximal response to the EGF stimulation was observed at a low concentration (0.1 ng per mL). When two different types of fibroblasts were treated with IL-1β or EGF, increased expression with different degrees and rates of three different HAS genes and subsequent increased synthesis of hyaluronan were also observed. In addition, HAS1 gene expression was not detectable in the mucosal fibroblasts, while weak HAS3 gene expression was detected in the dermal fibroblasts. Taken together, it is likely that the regulation of the expression of the three different HAS genes is different between oral mucosa and skin, which may be of significance for elucidating some of the differences between these tissues in wound healing. cultured human oral mucosal epithelial cells epidermal growth factor hyaluronan human hyaluronan synthase interleukin-1β open reading frame polymerase chain reaction reverse transcriptase-polymerase The process of wound healing depends upon a variety of interactions between cells and the extracellular matrix (Clark and Henson, 1988Clark R.A.F. Henson P.M. The Molecular and Cellular Biology of Wound Repair. Plenum Press, New York1988: 3-33Crossref Google Scholar). It is well known that hyaluronan not only supports tissue architecture as a passive structural component of the matrix in various connective tissues but is also involved in dynamic cellular processes such as cell migration and cell–cell recognition during wound healing and inflammation (Weigel et al., 1997Weigel P.H. Hascall V.C. Tammi M. Hyaluronan synthases.J Biol Chem. 1997; 272: 13997-14000Crossref PubMed Scopus (591) Google Scholar;Knudson et al., 1989Knudson W. Biswas C. Li X.-Q. Nemec R.E. Toole B.P. The biology of hyaluronan.in: Evered D. Whelan J. Ciba Foundation Symposium. Vol. 143. Wiley, Chichester, UK1989: 150-169Google Scholar;Evered et al., 1989Turley E.A. The biology of hyaluronan.in: Evered D. Whelan J. Ciba Foundation Symposium. Vol. 143. Wiley, Chichester, UK1989: 121-137Google Scholar). Three different mammalian hyaluronan synthases, HAS1, HAS2, and HAS3, have been identified and characterized (Rosa et al., 1988Rosa F. Sargent T.D. Rebbert M.L. et al.Accumulation and decay of DG42 gene products follow a gradient pattern during Xenopus embryogenesis.Dev Biol. 1988; 129: 114-123Crossref PubMed Scopus (84) Google Scholar;Itano and Kimata, 1996aItano N. Kimata K. Expression cloning and molecular characterization of HAS protein, a eukaryotic hyaluranan synthase.J Biol Chem. 1996; 271: 9875-9878Crossref PubMed Scopus (152) Google Scholar, Itano and Kimata, 1996bItano N. Kimata K. Molecular cloning of human hyaluronan synthase.Biochem Biophys Res Commun. 1996; 222: 816-820Crossref PubMed Scopus (98) Google Scholar; Shyjan et al., 1996Shyjan A. Heldin P. Butcher E. Yoshino T. Briskin M. Functional cloning of the cDNA for a human hyaluronan synthase.J Biol Chem. 1996; 271: 23395-23399Crossref PubMed Scopus (141) Google Scholar; Spicer et al., 1996Spicer A.P. Augustine M.L. Mcdonald J.A. Molecular cloning and characterization of putative mouse hyaluronan synthase.J Biol Chem. 1996; 271: 23400-23406Crossref PubMed Scopus (149) Google Scholar; Watanabe and Yamaguchi, 1996Watanabe K. Yamaguchi Y. Molecular identification of a putative human hyaluronan synthase.J Biol Chem. 1996; 271: 22945-22948Crossref PubMed Scopus (178) Google Scholar; Spicer et al., 1997Spicer A.P. Olson J.S. McDonald J.A. Molecular cloning and characterization of a cDNA encoding the third putative mammalian hyaluronan synthase.J Biol Chem. 1997; 272: 8957-8961Crossref PubMed Scopus (150) Google Scholar). The three HAS genes show distinct expression patterns (Spicer and Mcdonald, 1998Spicer A.P. Mcdonald J.A. Characterization and molecular evolution of a vertebrate hyaluronan synthase gene family.J Biol Chem. 1998; 273: 1923-1932Crossref PubMed Scopus (279) Google Scholar) and the synthases are significantly different in their enzymatic properties and in their role in the pericellular hyaluronan coat formation (Itano et al., 1999Itano N. Sawai T. Yamada Y. et al.Three isoforms of mammalian hyaluronan synthase have distinct enzymatic properties.J Biol Chem. 1999; 274: 25085-25092Crossref PubMed Scopus (658) Google Scholar). The precise regulatory mechanism of the expression of each HAS is still unknown. It has been shown that the synthesis of hyaluronan is stimulated by some growth regulatory factors and anti-inflammatory cytokines such as epidermal growth factor (EGF) and IL-1β (Heldin et al., 1989Heldin P. Laurent T.C. Heldin C.H. Effect of growth factors on hyaluronan synthesis in cultured human fibroblasts.Biochem J. 1989; 258: 919-922Crossref PubMed Scopus (256) Google Scholar;Yung et al., 1996Yung S. Coles G.A. Davies M. IL-1 beta, major stimulator of hyaluronan synthesis I in vitro of human peritoneal cells: Relevance to peritonitis in CAPD.Kidney Int. 1996; 50: 1337-1343Crossref PubMed Scopus (64) Google Scholar;Kaback and Smith, 1999Kaback L.A. Smith T.J. Expression of hyaluranan synthase messenger ribonucleic acids and their induction by interleukin-1 beta in human orbital fibroblasts: Potential insight into the molecular pathogenesis of thyroid-associated ophthalmopathy.J Clin Endocrinol Metab. 1999; 84: 4079-4084Crossref PubMed Google Scholar), which have also been shown to be agents that promote wound healing (Brown et al., 1986Brown G.L. Curtsinger L. Brightwell J.R. et al.Enhancement of epidermal regeneration by biosynthetic epidermal growth factor.J Exp Med. 1986; 163: 1319-1324Crossref PubMed Scopus (244) Google Scholar;Gailit et al., 1994Gailit J. Welch M.P. Clark R.A. TGF-beta 1 stimulates expression of keratinocyte integrins during re-epithelialization of cutaneous wounds.J Invest Dermatol. 1994; 103: 221-227Crossref PubMed Scopus (191) Google Scholar). Considering the above findings on the existence of three different hyaluronan synthases, it is likely that the expression of each hyaluronan synthase is regulated in a different manner, depending on the difference of growth regulatory factors or cytokines. Langer and Vacanti, 1993Langer R. Vacanti J.P. Tissue engineering.Science. 1993; 260: 920-926Crossref PubMed Scopus (8665) Google Scholar described a new technology for solid organ transplants called tissue engineering, which involves the morphogenesis of new tissue using constructs formed from isolated cells cultured with growth regulatory factors and biocompatible scaffolds.Ueda et al., 1995Ueda M. Hata K. Horie K. Torii S. The potential of oral mucosal cells for cultured epithelium: A preliminary report.Ann Plast Surg. 1995; 35: 498-504Crossref PubMed Scopus (52) Google Scholar fabricated cultured epithelium sheets for skin repair using cultured human oral mucosal epithelial (COME) cells and attained good clinical results. Therefore, we focused on the relationship among the growth regulatory factors, extracellular matrix, and epidermal and dermal cells, with the aim of tissue regeneration without scarring. Most skin lesions heal rapidly and efficiently within a week or two; however, scars remain where the collagen matrix has been poorly reconstituted. Oral mucosa, on the other hand, rarely suffers from scarring in the process of wound healing (Tsai et al., 1995Tsai C.Y. Hata K. Trii S. Matsuyama M. Ueda M. Contraction potency of hypertrophic scar-derived fibroblasts in a connective tissue model: In vivo analysis of wound contraction.Ann Plast Surg. 1995; 35: 638-646Crossref PubMed Scopus (18) Google Scholar) and appears to be different from skin in this regard. We have developed a method to fabricate cultured epithelium for skin repair using COME cells, which are a potential new source of cells for cultured epithelial grafts without scarring (Ueda et al., 1995Ueda M. Hata K. Horie K. Torii S. The potential of oral mucosal cells for cultured epithelium: A preliminary report.Ann Plast Surg. 1995; 35: 498-504Crossref PubMed Scopus (52) Google Scholar). Therefore, comparison of the regulation of hyaluronan synthesis in oral mucosa with that in skin may yield important insights into the wound-healing processes characteristic of oral mucosa lesions. Histological analysis of the hyaluronan distribution in skin showed that this molecule is localized not only in the dermis but also in the epidermis (Tammi et al, 1984). The ability of keratinocytes to synthesize hyaluronan has been reported in both cell cultures (Brown and Parkinson, 1983Brown K.W. Parkinson Glycoproteins and glycosaminoglycans of cultured normal human epidermal keratinocytes.J Cell Sci. 1983; 61: 325-338PubMed Google Scholar) and organ cultures (Tammi et al., 1989Tammi R. Ripellino J.A. Margolis R.U. Maibach H.I. Tammi M. Hyaluronate accumulation in human epidermis treated with retinoic acid in skin.J Invest Dermatol. 1989; 92: 326-332Abstract Full Text PDF PubMed Scopus (117) Google Scholar;Agren et al., 1995Agren U.M. Tammi M. Tammi R. Hydrocortisone regulation of hyaluronan metabolism in human skin organ culture.J Cell Physiol. 1995; 164: 240-248Crossref PubMed Scopus (35) Google Scholar). Skin grafts have been shown to promote wound closure by releasing a variety of cytokines (Krejci et al., 1991Krejci N.C. Cuono C.B. Langdon R.C. In vitro reconstitution of skin: Fibroblasts facilitate keratinocyte growth and differentiation on acellular reticular dermis.J Invest Dermatol. 1991; 97: 843-848Abstract Full Text PDF PubMed Google Scholar;Martin, 1997Martin P. Wound healing—Aiming for perfect skin regeneration.Science. 1997; 276: 75-81Crossref PubMed Scopus (3430) Google Scholar). Therefore, it is likely that the expression of three different hyaluronan synthases in the skin may be regulated by these factors. Actually,Sugiyama et al., 1998Sugiyama Y. Shimada A. Sayo T. Sakai S. Inoue S.J. Putative hyaluronan synthase mRNA are expressed in mouse skin and TGF-beta upregulates their expression in cultured human skin cells.J Invest Dermatol. 1998; 110: 116-121Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar found that TGF-β upregulates HAS 1 and HAS2 expression independently in cultured human skin fibroblasts. Although HAS1, 2, and 3 mRNAs have been detected in various tissues by northern blot analyses (Spicer and Mcdonald, 1998Spicer A.P. Mcdonald J.A. Characterization and molecular evolution of a vertebrate hyaluronan synthase gene family.J Biol Chem. 1998; 273: 1923-1932Crossref PubMed Scopus (279) Google Scholar), comparative studies on the distributions of HAS1, 2, and 3 expression as well as on the effect of cytokines on their expression between oral mucosa and skin have not yet been performed. Real-time RT-PCR analysis enables one to detect quantitatively certain mRNAs in RNA samples, although careful attention must be paid to establishing suitable PCR conditions and primers (Gibson et al., 1996Gibson U.E.M. Heid C.A. Williams A. Novel method for real time quantitative RT-PCR.Genome Res. 1996; 6: 995-1001Crossref PubMed Scopus (1731) Google Scholar;Heid et al., 1996Heid C.A. Stevens J. Williams P.M. Real time quantitative PCR.Genome Res. 1996; 6: 986-994Crossref PubMed Scopus (4817) Google Scholar). In this study, we took advantage of this new method and also developed culture systems to assess the expression of the HAS1, HAS2, and HAS3 genes and hyaluronan synthesis in COME cells, oral mucosal fibroblasts, and skin dermal fibroblasts, and compared them before and after simulation by IL-1β or EGF. The current results may help to clarify some of the mechanisms of the different responses in wound healing between oral mucosa and skin, and define potential targets for specific therapy directed at modulating hyaluronan synthesis in tissue engineering. Using real-time RT-PCR analysis, we measured the absolute amounts of the mRNAs of the three different hyaluronan synthases, HAS 1, HAS2, and HAS3, in the COME cells. Comparisons of the expression among different samples by normalizing each of the absolute amounts enabled us to elucidate how the addition of IL-1β or EGF affected the expression of the HAS 1, HAS2, and HAS3 genes. Treatment of COME cells with 0.1–100 ng per mL of IL-1β or EGF for 3 h resulted in a marked increase in both HAS1 and HAS2 mRNAs (Figure 1 and Figure 2). Maximal levels of HAS1 and HAS2 expression were attained at 100 ng per mL IL-1β and 0.1 ng per mL EGF, respectively. Since EGF at this concentration (0.1 ng per mL) is used as one of the supplements for HuMedia-KG2 medium (commercially available), our finding indicates that this medium is suitable at least for hyaluronan synthesis by COME cells. The amounts of HAS1 and HAS2 mRNAs were increased in a concentration-dependent manner by IL-1β at all concentrations tested, while those in EGF-treated COME cells were maximal at 0.1 ng per mL EGF and decreased to almost the basal level at the higher concentrations of EGF in order of HAS1 and HAS expression Figure 3. A much smaller increase of HAS3 than of HAS1 and HAS2 expression was observed in the early phase of the stimulation (Figure 1 and Figure 2). The concentration-dependent changes in HAS3 expression after IL-1β or EGF stimulation were similar to, but less marked than, those in HAS1 and HAS2 expression Figure 3.Figure 2Stimulation by EGF of HAS1 (a), HAS2 (b), and HAS3 (c) gene expression in COME cells. COME cells were cultured in the presence of EGF at the indicated concentrations: 0 ng per mL (▴), 0.1 ng per mL (•), 1 ng per mL (▵), 10 ng per mL (×), and 100 ng per mL (○). After 3, 6, 12, and 24 h, cells were lysed and total RNA were extracted. Total RNA were also extracted from the cells just before the addition of EGF as a control (0 h). The ordinate represents the expression coefficient for each mRNA, which was calculated as described in Figure 1. Each point is the mean value obtained from five independent experiments in which the differences were less than 10%. *Significantly different from the control value; p<0.01. Boxed values are also significantly different (p<0.01) from the control.View Large Image Figure ViewerDownload (PPT)Figure 3Comparison of concentration-dependent changes of HAS1 (a), HAS2 (b), and HAS3 (c) gene expression between stimulation by IL-1β or EGF. COME cells were cultured in the presence of the indicated concentrations of 0.1–100 ng per mL IL-1β (▾) or EGF (▿). After 3 h, cells were lysed and total RNA were extracted. Total RNA were also extracted from the cells just before the treatment for the 0 h sample. Quantitation of each mRNA (HAS1, HAS2, and HAS3) was performed using equal amounts of total RNA (200 ng) by real-time RT-PCR as described in Materials and Methods. The ordinate represents the expression coefficient for each mRNA, which was calculated as described in Figure 1. Each point is the mean value obtained from five independent experiments in which the differences were less than 10%. *Significantly different from the control value; p<0.01.View Large Image Figure ViewerDownload (PPT) Comparisons of expression coefficients at 3 h, when the maximal stimulation was observed, suggested that the absolute amount of HAS2 mRNA was about 20-fold that of HAS1 mRNA, and about 8-fold that of HAS3 mRNA in COME cells (Figure 1, Figure 2 and Figure 3). Therefore, the HAS2 mRNA appeared to be responsible for most of the hyaluronan synthase expression in the early phase of IL-1β or EGF stimulation. The proportion of HAS3 mRNA, however, appeared to become more significant in the late phase of the stimulation (Figure 1 and Figure 2). We examined the effects of IL-1β or EGF on the expression of HAS1, HAS2, and HAS3 mRNAs in cultured oral mucosal fibroblasts and dermal fibroblasts using the same method. Stimulation of these fibroblasts with 0.1–100 ng per mL IL-1β or EGF for 3 h resulted in a marked increase of HAS1 mRNA in cultured dermal fibroblasts, HAS2 mRNA in both dermal and cultured oral mucosal fibroblasts, and HAS3 mRNA in cultured oral mucosal fibroblasts. Maximal levels of HAS1 and HAS2 expression were attained at 1 ng per mL IL-1β in dermal fibroblasts, while those of HAS2 and HAS3 expression were attained at 1 ng per mL EGF in oral mucosal fibroblasts Figure 4. Neither significant HAS1 gene expression in cultured oral mucosal fibroblasts nor HAS3 gene expression in dermal fibroblasts was detected at any concentration of EGF or IL-1β Figure 4. Interestingly, lower expression of the HAS2 gene was observed in both types of fibroblasts after EGF stimulation compared with that after IL-1β stimulation, whereas the opposite was observed in COME cells after EGF and IL-1β stimulation (compare concentration-dependency in Figure 4 with that in Figure 3). We then investigated the upregulation of the expression of HAS1, HAS2, and HAS3 mRNAs when oral mucosal and dermal fibroblasts were stimulated with 1 ng per mL IL-1β or EGF for different periods. Treatment of oral mucosal fibroblasts with IL-1β or EGF resulted in marked and moderate increases, respectively, in the amount of HAS2 mRNA, with maximal stimulations at 6 and 3 h, respectively (Figure 5 and Figure 6). On the other hand, the amount of HAS3 mRNA in mucosal fibroblasts was increased time dependently for at least 24 h by IL-1β stimulation, but reached a plateau with a 2-fold increase at 3 h in response to EGF stimulation (Figure 5 and Figure 6). When dermal fibroblasts were treated with IL-1β or EGF, the upregulation of expression of HAS1 and HAS2 mRNAs was observed as early as 3 h after stimulation with either agent. The amount of HAS1 mRNA, however, was further increased time dependently up to 12 h, and then decreased to almost the basal level. The level of HAS2 mRNA was increased to a maximum at 6 h after IL-1β stimulation, decreased at 12 h, and again increased slightly thereafter. The maximal HAS2 expression level after IL-1β stimulation in dermal fibroblasts was 1.5 times higher than that after EGF stimulation (expression coefficient, 1.45 vs 0.93) (compare Figure 5 with Figure 6). The maximal stimulations by these cytokines of HAS1, HAS2, and HAS3 gene expression in both types of fibroblasts were mostly observed later than those in COME cells, as described above. The expression coefficients in Figure 5 and Figure 6 suggest that in both types of cells, HAS2 mRNA was more abundant (about 5-fold) than the other HAS mRNAs.Figure 6Comparison of stimulation by EGF of HAS1 (a), HAS2 (b), and HAS3 (c) gene expression between oral mucosal and dermal fibroblasts. Oral mucosal (▪) and dermal (□) fibroblasts were cultured in the presence of 1 ng per mL EGF. After 3, 6, 12, and 24 h, cells were lysed and total RNA were extracted. For controls (0 h sample), total RNA were also extracted from the cells just before the addition of EGF. Quantitation of each mRNA (HAS1, HAS2, and HAS3) was performed using equal amounts of total RNA (200 ng) by real-time RT-PCR as described in Materials and Methods. The ordinate represents the expression coefficient for each mRNA, which was calculated as described in Figure 1. Each point is the mean value obtained from five independent experiments in which the differences were less than 10%. *Significantly different from the control value; p<0.01.View Large Image Figure ViewerDownload (PPT) These differences suggest that HAS1, HAS2, and HAS3 gene expression in oral mucosal and dermal fibroblasts is regulated by IL-1β and EGF in distinct manners that are cell-origin-specific and cytokine-specific. To monitor the rate of hyaluronan synthesis at different times between 0 and 24 h after treatment with 1 ng per mL IL-1β or EGF, we measured the HA concentration in the culture medium at different times by a competitive ELISA-like assay, as shown in Figure 7. In COME cell cultures, IL-1β or EGF treatment induced an ∼2–7-fold increase of newly synthesized hyaluronan during the stimulation, compared to the level at 3 h Figure 7a. The amounts of HA in the cultures treated with either agent further increased time dependently up to 24 h. On the other hand, the amounts of HA in cultures of oral mucosal fibroblasts treated with IL-1β or EGF showed an ∼23–47-fold increase, and that in cultures of dermal fibroblasts showed a 10–20-fold increase, compared to the levels at 3 and 6 h Figure 7b, c. It is of note that neither oral mucosal fibroblasts nor dermal fibroblasts showed an IL-1β- or EGF-induced increase in hyaluronan synthesis until 6 h and that higher levels of HA were generated in oral fibroblasts than in dermal fibroblasts after IL-1β stimulation Figure 7b, whereas higher levels of HA were observed in dermal fibroblasts than in oral mucosal fibroblasts after EGF stimulation Figure 7c Overall, it was interesting to see that the increased expression levels of HAS after IL-1β or EGF stimulation resulted in increased rates of HA synthesis, although concentration dependency of the increase of HA synthesis by these agents remains to be determined. A major goal of wound-healing biology is to discover how skin can be induced to reconstruct damaged parts more perfectly (Martin, 1997Martin P. Wound healing—Aiming for perfect skin regeneration.Science. 1997; 276: 75-81Crossref PubMed Scopus (3430) Google Scholar). In this regard, we firstly noted the previous observation that oral mucosa rarely suffers from scarring in the process of wound healing and appears to be different from skin (Tsai et al., 1995Tsai C.Y. Hata K. Trii S. Matsuyama M. Ueda M. Contraction potency of hypertrophic scar-derived fibroblasts in a connective tissue model: In vivo analysis of wound contraction.Ann Plast Surg. 1995; 35: 638-646Crossref PubMed Scopus (18) Google Scholar), and therefore focused on differences in physiological responses to wound healing between oral mucosa cells and skin cells in this study. Wound healing involves many dynamic cellular processes, such as cell proliferation, cell migration, cell–cell interaction, and inflammation (Martin, 1997Martin P. Wound healing—Aiming for perfect skin regeneration.Science. 1997; 276: 75-81Crossref PubMed Scopus (3430) Google Scholar). It has been found that hyaluronan is deeply involved in these dynamic cellular processes during wound healing and inflammation (Knudson et al., 1989Knudson W. Biswas C. Li X.-Q. Nemec R.E. Toole B.P. The biology of hyaluronan.in: Evered D. Whelan J. Ciba Foundation Symposium. Vol. 143. Wiley, Chichester, UK1989: 150-169Google Scholar;Evered et al., 1989Turley E.A. The biology of hyaluronan.in: Evered D. Whelan J. Ciba Foundation Symposium. Vol. 143. Wiley, Chichester, UK1989: 121-137Google Scholar;Weigel et al., 1997Weigel P.H. Hascall V.C. Tammi M. Hyaluronan synthases.J Biol Chem. 1997; 272: 13997-14000Crossref PubMed Scopus (591) Google Scholar). It is also known that some cell growth factors and cytokines facilitate wound healing and re-epithelialization by stimulating keratinocyte proliferation and migration (Clark and Henson, 1988Clark R.A.F. Henson P.M. The Molecular and Cellular Biology of Wound Repair. Plenum Press, New York1988: 3-33Crossref Google Scholar;Gailit et al., 1994Gailit J. Welch M.P. Clark R.A. TGF-beta 1 stimulates expression of keratinocyte integrins during re-epithelialization of cutaneous wounds.J Invest Dermatol. 1994; 103: 221-227Crossref PubMed Scopus (191) Google Scholar;Hubner et al., 1996Hubner G. Brauchle M. Smola H. et al.Differential regulation of pro-inflammatory cytokines during wound healing in normal and glucocorticoid-treated mice.Cytokine. 1996; 8: 548-556Crossref PubMed Scopus (358) Google Scholar;Martin, 1997Martin P. Wound healing—Aiming for perfect skin regeneration.Science. 1997; 276: 75-81Crossref PubMed Scopus (3430) Google Scholar;Tammi and Tammi, 1998Tammi R. Tammi M. Glycoforum. 1998: 1-12http://www.glycoforum.gr.jp/Google Scholar;Pienimaki et al., 2001Pienimaki J.P. Rilla K. Fulop C. et al.Epidermal growth factor activates hyaluronan synthesis 2 in epidermal keratinocytes and increases pericellular and intracellular hyaluronan.J Biol Chem. 2001; 276: 20428-20435Crossref PubMed Scopus (157) Google Scholar). Therefore, in this study we examined the relationships between hyaluronan synthesis and cellular responses to two cell growth regulatory factors involved in wound healing of the skin, namely IL-1β and EGF. The present results demonstrated that the expression of the three different HAS genes was increased in COME cells by IL-1β or EGF treatment, and there was a corresponding increase of hyaluronan synthesis in these cells after the treatments. Therefore, such treatments may induce changes in the extracellular environment via the increased synthesis and accumulation of hyaluronan. The present data also showed that the stimulations varied with different concentrations and times of treatment with the cytokines and that the stimulation patterns were highly dependent upon cell origins. This may be important for understanding the difference in wound healing between oral mucosal epithelium and epidermis. Our present study did not focus on human keratinocytes, because they have already been studied in detail in a few studies, with the following results.Sugiyama et al., 1998Sugiyama Y. Shimada A. Sayo T. Sakai S. Inoue S.J. Putative hyaluronan synthase mRNA are expressed in mouse skin and TGF-beta upregulates their expression in cultured human skin cells.J Invest Dermatol. 1998; 110: 116-121Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar showed that human keratinocytes expressed HAS1 mRNA and a trace of HAS2 mRNA, and that when the culture was stimulated with TGF-β, HAS1 mRNA in keratinocytes was upregulated but HAS2 mRNA was not. A more recent study byPienimaki et al., 2001Pienimaki J.P. Rilla K. Fulop C. et al.Epidermal growth factor activates hyaluronan synthesis 2 in epidermal keratinocytes and increases pericellular and intracellular hyaluronan.J Biol Chem. 2001; 276: 20428-20435Crossref PubMed Scopus (157) Google Scholar showed that HAS2 mRNA was expressed in a rat keratinocyte cell line and EGF stimulation brought about an increase in HAS2 mRNA corresponding to about a 30-fold enhancement of hyaluronan production from the basal synthesis rate. They also showed that there was no increase in HAS1 or HAS3 in the cell line, but HAS2 mRNA increased 2–3-fold with less than 2 h following stimulation with EGF. Our present study on oral mucosal epithelium cells, however, demonstrated that in those cells the expression of HAS1 and HAS3 was upregulated after EGF stimulation, although these mRNAs were expressed at lower levels than HAS2 mRNA, and, in addition, HAS2 mRNA increased 2–11-fold depending on the concentration of the inducing agent Figure 3. This finding may have depended on our use of the different cells or of the real-time RT-PCR method, which is more accurate than the methods used in earlier studies. Our finding also suggests that HAS1 and HAS3 might have distinctive effects on the wound healing of oral mucosal epithelia. Sugiyama et al., 1998Sugiyama Y. Shimada A. Sayo T. Sakai S. Inoue S.J. Putative hyaluronan synthase mRNA are expressed in mouse skin and TGF-beta upregulates their expression in cultured human skin cells.J Invest Dermatol. 1998; 110: 116-121Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar demonstrated that when human dermal fibroblasts were stimulated with TGF-β, both HAS1 and HAS2 mRNAs were upregulated.Zhang et al., 2000Zhang W. Watson C.E. Liu C. Williams K.J. Werth V.P. Glucocorticoids induce a near-total suppression of hyaluronan synthase mRNA in dermal fibroblasts and in osteoblasts: A molecular mechanism contributing to organ atrophy.Biochem J. 2000; 349: 91-97Crossref PubMed Scopus (59) Google Scholar found by a quantitative study of HAS mRNA level
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