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Cell Biology International (2009) 33, 11441148 (Printed in Great Britain)
Calcitonin gene-related peptide increases proliferation of human HaCaT keratinocytes by activation of MAP kinases
Xiao‑Jing Yu, Chun‑Yang Li*, Yong‑Hao Xu, La‑Mei Chen and Chun‑Lei Zhou
Department of Dermatology, Qilu Hospital, University of Shandong, Jinan 250012, China
Psoriasis is a chronic disease characterized by keratinocyte hyperproliferation and inflammation. It has been demonstrated that the expression of calcitonin gene-related peptide (CGRP) is elevated in psoriasis lesions and CGRP-containing neuropeptide nerve fibers are denser in the psoriatic epidermis. CGRP has been previously described to influence proliferation of several cell types, such as Schwann cell, tracheal epithelial cells, and human gingival fibroblasts. In the present study, we determined the effect of CGRP on HaCaT keratinocyte proliferation and the role of mitogen-activated protein kinases (MAPKs) in CGRP induced keratinocyte proliferation. Our data indicate CGRP increased [3H]-thymidine incorporation and MTT activity of HaCaT in a concentration-dependent manner. CGRP also enhanced serum-induced HaCaT cell proliferation. HaCaT cells cultured with CGRP had a significant increase in phosphorylated ERK1/2, p38 and JNK, and CGRP induced DNA synthesis was inhibited by PD 98059 or SB 203580, selective inhibitors of MAP kinase kinase (MEK, which is upstream from ERK) and p38, respectively. These findings suggest that HaCaT cell proliferate in response to CGRP, which is mediated by phosphorylation of ERK1/2 and p38 MAPK.
Keywords: CGRP, Cell proliferation, ERK1/2, JNK, p38 MAPK, HaCaT keratinocytes, Psoriasis.
*Corresponding author. Tel.: +86 531 82169390; fax: +86 531 86927544.
Psoriasis is a chronic hyperproliferative skin disease characterized by keratinocyte hyperproliferation and inflammation (Baker and Fry, 1992; Creamer et al., 1997). Although dysfunction of the immune system is known to be an important factor in the pathogenesis of psoriasis, there is also strong evidence that keratinocyte hyperproliferation contribute to the disease. The proliferation and differentiation of keratinocytes are controlled by a complex network of growth factors and cytokines (Luger and Schwarz, 1990). Calcitonin gene-related peptide (CGRP), a 37-amino acid peptide, is one of the most abundant neuropeptides in human and rodent skin. It has been demonstrated that the expression of CGRP is elevated in psoriasis lesions and CGRP-containing neuropeptide nerve fibers are denser in the psoriatic epidermis (Jiang et al., 1998; Farber and Raychaudhuri, 1999). These findings suggest that CGRP may play a significant role in the pathophysiologic process of psoriasis.
The effects of CGRP on cell proliferation are more complex and context dependent. CGRP has been previously described to positively influence proliferation of Schwann cells (Cheng et al., 1995), tracheal epithelial cells (White et al., 1993), and human Gin-1 gingival fibroblasts (Kawase et al., 1999). On the contrary, it was reported to posses antiproliferative effects on cultured smooth muscle cells (SMC) (Deng et al., 2006; Qin et al., 2004; Chattergoon et al., 2005). The proliferation of several phenotypes of cells is mediated by mitogen-activated protein kinases (MAPKs), a family of serine-threonine protein. There are three well-characterized MAPK subfamilies in mammalian cells: extracellular-signal-regulated protein kinase (ERK), the p38 mitogen-activated protein kinases (p38 MAPKs) and the c-Jun N-terminal kinase (JNK) (Cowan and Storey, 2003). On activation by phosphorylation of both threonine and tyrosine residues, these kinases phosphorylate intracellular enzymes and transcription factors. The activation of MAPKs is a key component in signal transduction associated with cell proliferation.
This study was designed to investigate the effect of CGRP on cell proliferation by the human keratinocyte cell line HaCaT, and the role of MAPKs in CGRP induced HaCaT proliferation. We found that CGRP increased HaCaT proliferation and the proliferative effects were mediated by phosphorylation of ERK1/2 and p38.
2 Materials and methods
Human α-calcitonin gene-related peptide and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) were purchased from Sigma (Saint Louis, MO). PD 98059, SB 203580 and SP 600125 were obtained from Biosource (Camarillo, CA). [3H]-thymidine was purchased from Institute of High Energy Physics, Chinese Academy of Sciences (Beijing, China). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco (Carlsbad, CA). Mouse antiphospho-ERK1/2 (p-ERK1/2) antibody, rabbit anti-ERK1/2 antibody, mouse antiphospho-p38 (p-p38) antibody, mouse anti-p38 antibody, mouse antiphospho-JNK (p-JNK) antibody, mouse anti-JNK antibody and rabbit anti-actin antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
2.2 MTT and [3H]-thymidine incorporation assay for cell proliferation
Human keratinocyte cell lines HaCaT cells were cultured in DMEM supplemented with 10% fetal calf serum (FBS), 100 U/ml penicillin and 100
For [3H]-thymidine incorporation assay, 5
2.3 Western blot analysis
Stimulated cells were lysed with ice-cold lysis buffer containing 50
2.4 Statistical analysis
The statistical significance was calculated by the two-sample independent-groups t-test. p
3.1 CGRP increased HaCaT proliferation
In order to determine whether CGRP acts as a growth factor, cells were seeded in wells of a 96-well plate at a density of 3 Fig. 1 CGRP concentration-dependently increased proliferation of HaCaT keratinocytes. Confluent and growth-arrested HaCaT keratinocytes were incubated with various concentrations of CGRP in 0.2% FBS/DMEM for 24
CGRP concentration-dependently increased proliferation of HaCaT keratinocytes. Confluent and growth-arrested HaCaT keratinocytes were incubated with various concentrations of CGRP in 0.2% FBS/DMEM for 24
3.2 CGRP augmented serum-induced proliferation
Serum contains a variety of mitogenic substances. HaCaT cells can respond synergistically to a wide variety of mitogen combinations. CGRP may interact with these substances and affect HaCaT cell proliferation. To determine this, we treated confluent, serum-free HaCaT cells with 10% FBS in the absence or presence of CGRP (10
CGRP enhanced serum-induced proliferations of HaCaT keratinocytes. Confluent and growth-arrested HaCaT keratinocytes were treated with 10% FBS in the absence or presence of CGRP (10
3.3 Roles of MAPKs in CGRP induced proliferation
Next, we determined if MAPKs play any role in CGRP induced increase in proliferation. HaCaT cells were pretreated for one hour with PD 98059, SB 203580, or SP 600125, followed by 24-h CGRP treatment and [3H]-thymidine incorporation assay. The CGRP induced DNA synthesis of HaCaT cells was attenuated by the addition of PD 98059 or SB 203580, but not SP 600125 (Fig. 3).
Fig. 3 Effects of MAPKs inhibitors on CGRP induced increase of proliferation in HaCaT keratinocytes. Confluent and growth-arrested HaCaT keratinocytes were pretreated for 1
Effects of MAPKs inhibitors on CGRP induced increase of proliferation in HaCaT keratinocytes. Confluent and growth-arrested HaCaT keratinocytes were pretreated for 1
In order to demonstrate in direct evidence of the activation of MAPKs, HaCaT cells were treated with 10
CGRP increased expression of phosphorylated MAPKs in HaCaT keratinocytes. Confluent and growth-arrested HaCaT keratinocytes were incubated with 10
Furthermore, total and phosphorylated p38, ERK1/2, and JNK were determined using the cellular protein of HaCaT cells treated with CGRP for 24
Effects of MAPKs inhibitors on CGRP induced activation of MAPKs. Confluent and growth-arrested HaCaT keratinocytes were pretreated for 1
HaCaT cells are the product of an experimental line derived from normal epidermal keratinocytes; they are immoral, maintain differentiation potential and are used as a model of keratinocytes function (Boukamp et al., 1988). Although there are some reports on the different responses between normal primary keratinocytes and HaCaT (Weninger et al., 1998; Kwon et al., 2004; Park et al., 2005), HaCaT exhibits most of the characteristics of the basal keratinocytes (Wraight et al., 1994; Wraight and Werther, 1995).
In this study we have demonstrated that CGRP increases proliferation in serum-free condition and enhances serum-induced proliferation of confluent HaCaT cells. This observation is consistent with the reports that CGRP stimulate DNA synthesis in cultured keratinocytes and proliferation of a human squamous cell carcinoma cell line in vitro (Takahashi et al., 1993). These findings have important clinical significance, because over expression of CGRP was found in psoriasis (Jiang et al., 1998; Farber and Raychaudhuri, 1999). Most recently, it was found that CGRP contributes to the UVB-induced keratinocytes proliferation and acanthosis in UVB-irradiated murine skin (Seike et al., 2002). Moreover, we found that CGRP enhances serum-induced HaCaT cell proliferation that occurs in epidermis of psoriasis where there is inflammation leading to increase in cytokines mitogen for keratinocytes. The findings suggest that the mitogenic effects of the cytokines would be enhanced by CGRP, and augment the HaCaT cell hyperplasia.
Phosphorylation of ERK1/2 has been reported to mediate mitogen-induced proliferation, while the phosphorylation of p38 and JNK are activated by a variety of non-specific stimuli such as changes in oxidation, osmolarity, and inflammatory cytokines (Johnson and Lapadat, 2002; Kyriakis and Avruch, 1996). The important roles of MAPKs activation in keratinocyte proliferation induced by IL-6, hepatocyte growth factor, EGF, estradiol and so on have been reported (Gallucci et al., 2004; Yano et al., 2003; Delehedde et al., 2002; Kaufmann and Thiel, 2002; Verdier-Sevrain et al., 2004). However, it is not known if MAPKs mediate CGRP induced HaCaT cell proliferation. In this study, for the first time, we have demonstrated that CGRP induced proliferation of HaCaT cells is associated with MAPKs. Since the inhibitors of ERK1/2 and p38 blocked CGRP induced proliferation, our data suggest that the activation of ERK1/2 and p38 is important for the CGRP induced increase in HaCaT cell proliferation. In our study, there are some differences in the time required for activation of MAPKs after CGRP stimulation amongst the MAPKs. P38 and JNK were rapidly activated by CGRP, which was as early as 5
In conclusion, our results demonstrate that CGRP increases HaCaT cells proliferation, and also enhances serum-induced HaCaT cells proliferation. In addition, the activation of ERK1/2 and p38 play an important role in mediating the CGRP induced proliferation by HaCaT keratinocytes. These findings suggest that CGRP which is elevated expressed in epidermis of psoriasis may involve in the pathogenesis of psoriasis by enhancing keratinocyte proliferation.
This work was supported by Natural Science Grant (Y2005C67) from the Shandong Ministry of Science and Technology.
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Received 27 July 2009; accepted 28 July 2009doi:10.1016/j.cellbi.2009.07.003