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Intracellular signaling molecules involved in vasoactive intestinal peptide-mediated wound healing in human bronchial epithelial cells
peptides 28 (2007) 1667–1673
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/peptides
Intracellular signaling molecules involved in vasoactive intestinal peptide-mediated wound healing in human bronchial epithelial cells Cha Xiang Guan a,*, Yan Ru Cui a, Min Zhang a, Hong Bo Bai a, Ravitej Khunkhun b, Xiang Fang b a b
Department of Physiology, Central South University Xiangya Medical School, Changsha, Hunan 410078, China Department of Medicine, Harbor Hospital Center, Baltimore, MD 21225, USA
article info
abstract
Article history:
Vasoactive intestinal peptide (VIP), a non-adrenergic, non-cholinergic neuromediator, plays
Received 14 February 2007
an important role in maintaining the bronchial tone of the airway and has anti-inflamma-
Received in revised form
tory properties. Recently, we reported that VIP enhances wound repair in human bronchial
14 July 2007
epithelial cells (HBEC). In the present study, we have identified the intracellular signaling
Accepted 17 July 2007
molecules that are involved in VIP-mediated wound healing in HBEC. The effects of VIP on
Published on line 1 August 2007
wound repair of HBEC were partially blocked by H-7 (a protein kinase C (PKC) inhibitor), W-7 (a calmodulin inhibitor), H-89 (a protein kinase A (PKA) inhibitor), and PD98059 (a specific
Keywords:
extracellular signal-regulated kinase (ERK) inhibitor). VIP-induced chemotactic migration
Vasoactive intestinal peptide
was inhibited in the presence of W-7, H-89, PD98059 or H-7. H-7, W-7, and H-89 were also
Human bronchial epithelial cells
found to decrease VIP-induced expression of Ki67 as well as the proliferation index in HBEC.
Wound healing
Furthermore, H-7, W-7, H-89, and PD98059 inhibited the expression of E-cd protein and
PKA
mRNA induced by VIP. These results suggest that intracellular signaling molecules such as
PKC
PKA, PKC, ERK, and calmodulin play important role in VIP-mediated wound healing of HBEC.
Calmodulin
# 2007 Elsevier Inc. All rights reserved.
ERK
1.
Introduction
Vasoactive intestinal peptide (VIP) is an important neuropeptide in the respiratory airway. VIP has many biological activities such as anti-inflammation, relaxation of airway smooth muscles, and inhibition of airway smooth muscle proliferation. VIP exerts its biological activities through the activation of VIP receptors. So far, three VIP receptors (VPAC1,
VPAC2, and PAC1) have been identified. These receptors share a common molecular architecture, which consists of seven transmembrane domains (7TM), three extracellular loops (EC1, EC2, and EC3), three intracellular loops (IC1, IC2, and IC3), a long amino-terminal extracellular domain, and an intracellular carboxyl terminus. There are four main intracellular signal pathways involved in VIP receptor-mediated intracellular signal transduction: (1) induction of adenylate
* Corresponding author. Tel.: +86 731 8836883; fax: +86 731 8830992. E-mail address: [email protected] (Cha Xiang Guan). Abbreviations: VIP, vasoactive intestinal peptide; HBEC, human bronchial epithelial cells; H-7, 1-(5-isoquinolinesulfonyl)-2-methylpi perazine; H-89, dihydrochloride; W-7, N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamid; SPF, S-phase fraction; PD98059, 2-amino-3methoxyflavon; Ki67, proliferating cell nuclear antigen; PI, proliferation index; GAPDH, glyceraldehyde phosphate dehydrogenase; PKA, protein kinase A; PKC, protein kinase C; MAPK, mitogen activated protein kinase; CaM, calmodulin 0196-9781/$ – see front matter # 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2007.07.027
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peptides 28 (2007) 1667–1673
cyclase, and consequently activation of cAMP-dependent protein kinase A (PKA) [2,8,10,13]; (2) activation of protein kinase C (PKC) [16,19]; (3) activation of calmodulin (CaM) [16,18]; (4) phosphorylation of cAMP-dependent mitogen activated protein kinase (MAPK) [4]. Previously, we reported that VIP accelerates wound healing in HBEC. It increases migration and proliferation of HBEC as well as expression of E-cadherin. These effects are blocked by a VIP receptor antagonist [6]. However, it is not known whether VIP receptor-mediated downstream signaling molecules are involved in its protective effects. In this study, we have determined the role of VIP receptors associated with downstream intracellular signaling molecules in the VIPmediated repair processes.
2.
Materials and methods
2.1.
Materials and cell culture
The HBEC line, 16HBE14o- was kindly provided by Dr. Gruenert from the University of California, San Francisco. DMEM/F12 mediums, H-7, W-7, H-89, PD98059, and VIP were purchased from Sigma Chemical (St. Louis, MO, USA). Trypase was from Amresco Company (Ohio, USA). Polycarbonate chemotaxis membrane was obtained from Neclepore Biotech (NJ, USA). DEPC was from BBI (Halifax, NS, Canada). Reverse transcription kits, Taq DNA polymerase, dNTP were from MBI (Ohio, USA). Trizol was from Invitrogen Life Technologies (California, USA). Primer was from Takara (Dalian, China). A DNA marker was from Beijing DingGuo Biotech (Beijing, China). The monoclonal antibodies against human E-cadherin, Ki67, and the secondary antibody were purchased from Beijing Zhongshan Biotech (Beijing, China). DAB/H2O2 coloring reagent was from Wuhan Boster Biological Technology (Wuhan, China). HBEC were cultured in a mixture medium of DMEM: F12 (1:1) containing 100 units/ml of penicillin, 100 units/ml of streptomycin, and 10% bovine calf serum and maintained at 37 8C in a humidified environment with 5% CO2.
2.2.
Wound healing assay
HBEC were cultured in 12-well plates with DMEM: F12 (1:1), and a small wound area was made in the confluent monolayer by mechanical scraping [1]. The wound area from each cultures was measured every 4 h up to 24 h. A linear regression equation of the remaining wound area to time was obtained. The repair index (RI), which is equal to the absolute value of slope, was used to identify the repair speed of HBEC.
2.3.
HBEC chemotaxis
HBEC chemotaxis was assessed by the blind well chamber technique. The confluent HBEC were treated with 0.05% trypsin at 37 8C for 10 min, and then 2.5% bovine calf serum was added to terminate the digestion. HBEC were then collected, and an aliquot of 200 ml HBEC (1.0 106 cells/ml) was loaded into the upper well of the chamber, while VIP or inhibitor was placed in the bottom chamber. The chambers were incubated for another 6 h. After incubation, the cells on
the top of the filter were removed by scraping. The filters were then fixed and stained with W & G stain. The number of cells remaining in the filters was counted with a microscope [6].
2.4.
Immunohistochemistry of Ki67
The cells were fixed with 95% ethanol and 0.1% Triton-X100. The fixed slides were incubated with 3% H2O2, and blocked with a normal goat serum for 20 min. The slides were then incubated with a mouse anti-human Ki67 monoclonal antibody (1:200) at 4 8C for overnight. The secondary antibody was added to the incubation at 37 8C for 30 min, and S-A/HRP was then added to the incubation at 37 8C for another 30 min. The slides were visualized with DAB/H2O2, counterstained with hematoxylin, and differentiated by chlorhydric acid alcohol. The slides were finally mounted with glycerol. The Ki67positive nuclei were evaluated by microscopy. The Ki67positive nuclei were counted through a minimum of 200 cells per field in five different fields. The Ki67 labeling index (Ki67 LI) is the number (%) of positive cells [17].
2.5.
Determination of cell cycling by flow cytometry
The cells were incubated with VIP in the presence or absence of inhibitors for 24 h, and were fixed in 70% ethanol at 4 8C for another 1 h. The cells were then incubated with RNase (100 mg/ ml, 1 ml) at 37 8C for 30 min. The cells were stained with 0.01% propidium iodide, filtered by grit for 20 min. The distribution of cells in the cell cycle was measured by flow cytometry. Adopted S-phase fraction (SPF) and the proliferation index (PI) were used to determine the proliferation of HBEC [6].
2.6.
Immunohistochemistry of E-cadherin
HBEC were fixed, and incubated with a mouse anti-human Ecadherin monoclonal antibody (1:200). After being mounted with glycerol, the slides were observed via microscope. The buffy granules were considered as E-cadherin product. Pictures were taken at 400 magnification, and background gray scales of those pictures were rectified by an Image-Pro Plus 5.0 image analytical system. The average gray scale was analyzed according to the instructions, and the higher the average gray scale value, the higher the expression of positive granules ratio [6].
2.7.
E-cadherin mRNA
The expression of E-cadherin was measured by RT-PCR, and the GAPDH mRNA expression was chosen as a standard. Total RNA was isolated, and 1 mg RNA was reverse transcribed in a 20 ml reaction buffer containing 4 ml 25 mM MgCl2, 2 ml 10 buffer, 0.5 ml RNasin, 15 U avian myeloblastosis virus, 0.5 mg oligo (dT) primer. The primer sequences were as follows: Ecadherin (502 bp) forward: 50 -TCC CAT CAG CTG CCC AGA AA30 , and reverse: 50 -TGA CTC CTG TGT TCC TGT TA-30 ; GAPDH (240 bp) forward: 50 -TGA TGA CAT CAA GAA GGT GGT GAA G30 , and reverse: 50 -TCC TTG GAG GCC ATG TGG GCC AT-30 [11,12]. PCR reaction conditions were as follows: 95 8C for 5 min, 94 8C for 1 min, 58 8C for 2 min, 72 8C for 3 min for 30
peptides 28 (2007) 1667–1673
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Fig. 1 – Effects of H-7 (10S5 M), H-89 (10S5 M), W-7 (10S5 M), and PD98059 (10S5 M) on closure of HBEC monolayer wounds induced by VIP (10S8 M). The HBEC monolayer was recorded by video microscopy, and the wound area from each culture was calculated by an Image Pro+ System. The remaining wound area/wound area at 0 h (%) of the groups in different time is shown in the top panel, while the RI is shown in the bottom panel. Data are represented as mean W S.E. from three separate experiments. **P < 0.01 vs. control group, ##P < 0.01 vs. VIP group.
cycles, then 72 8C for 5 min. The products were electrophoresed on a 2% agarose gel. The gel was then visualized by ultraviolet light, and the density of E-cadherin mRNA was measured [6].
Fig. 3 – Effects of H-7 (10S5 M), H-89 (10S5 M), W-7 (10S5 M), and PD98059 (10S5 M) on VIP-induced proliferation of HCEC. (A) The presence of Ki67 from each culture was determined by immunochemical method as described in Section 2. Ki67-positive nuclei were stained brown, while the Ki67-negtive nuclei were stained light blue. HBEC cultures were incubated with H-7, H-89, W-7, or PD98059 in the presence of VIP for 24 h. (B) The expression of Ki67 was expressed as the positive cell-labeling index (LI). Data are expressed as mean W S.E. from five separate experiments. **P < 0.01 vs. control, ##P < 0.01 vs. VIP groups.
2.8.
Statistical analyses
The data were expressed by mean S.E., and the difference between the multiple treatment groups was analyzed by ANOVA. P < 0.05 was considered as statistically significant.
Fig. 2 – Effects of W-7 (10S5 M), H-7 (10S5 M), H-89 (10S5 M), and PD98059 (10S5 M) on VIP-mediated HBEC chemotaxis. HBEC cultures were incubated with W-7, H-7, H-89, and PD98059 in the presence of VIP (10S7 M) for 6 h, and the chemotaxis was measured by the blind well chamber technique. Data values are mean W S.E. obtained from six separate experiments. **P < 0.01 vs. control group, ## P < 0.01 vs. VIP group.
3.
Results
3.1. The effect of H-7, W-7, PD98059, H-89 on VIP-mediated wound healing A small wound area in cultured HBEC was generated in each well with an area of approximately 0.832 0.104 mm2 at the beginning as described previously [6]. Consistent with our
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peptides 28 (2007) 1667–1673
previous findings, VIP significantly improved wound healing in a time-dependent manner. As indicated by RI, protective effects of VIP against mechanical scraping were partially blocked by pretreatment with H-7 (105 M, a PKC inhibitor), W7 (105 M, a calmodulin inhibitor), H-89 (105 M, a PKA inhibitor), and PD98059 (105 M, a specific ERK inhibitor) (Fig. 1). However, pretreatment with H-7, W-7, H-89 or PD98059 in the absence of VIP had no effect on wound healing of HBEC as compared with control (data not shown).
3.2. Effects of H-7, W-7, H-89, and PD98059 on VIP-induced chemotaxis We next determined the effect of VIP on HBEC migration. VIP (107 M) significantly accelerated the chemotactic migration, and the effects of VIP were decreased by pretreatment with W7, H-7, H-89 or PD98059 (the chemotactic intensities: 21.7 3.8 (VIP 107 M), 9.3 1.8 (VIP + W-7 105 M), 11.5 1.6 (VIP + H-7 105 M), 9.8 1.2 (VIP + H-89 105 M), and 11.8 1.2 (VIP + PD98059 107 M) cells/10 hpf) (Fig. 2).
3.3. Effects of H-7, W-7, H-89, and PD98059 on VIP-mediated proliferation of HBEC We first determined whether H-7, W-7, H-89, and PD98059 alone could affect the proliferation of HBEC using Ki67positive cells as an index for cell proliferation. The positive cell-labeling index (LI) in the control group was 9.1 0.9, and H-7, W-7, and PD 98059 alone did not change the Ki67-positive cell-LI (date not shown). VIP (108 M) increased the Ki67positive cell-LI to 12.4 0.9 after a 24 h of incubation (P < 0.01 versus control). H-7 (105 M), W-7 (105 M), H-89 (105 M), and PD98059 (105 M) could partially block VIP-induced Ki67 expression in HBEC (Fig. 3A and B). Furthermore, flow cytometry analysis of HBEC indicated that SPF and G2/M-phase fraction were increased in the
Fig. 4 – Effects of H-7, H-89, W-7, and PD98059 on VIPinduced changes of cell cycling. The HBEC cultures were incubated with vehicle (control) H-7 (10S5 M), H-89 (10S5 M), W-7 (10S5 M), and PD-98059 (10S5 M) in the presence of VIP (10S8 M). After incubation, the cell cycling was analyzed by flow cytometry. Data are expressed as mean W S.E. from three separate experiments, *P < 0.05 vs. control group, # P < 0.05 vs. VIP group, ##P < 0.05 vs. VIP group.
Fig. 5 – Effects of H-7 (10S5 M), H-89 (10S5 M), W-7 (10S5 M), and PD98059 (10S5 M) on the expression of E-cadherin protein. (A) HBEC cultures were incubated with different treatments for 28 h, plasmalemma-associated E-cadherin was then detected by a specific antibody against to Ecadherin. Monolayer images were recorded, and the average intensity was calculated by an Image Pro+ system. (B) Data are mean W S.E. from six separate experiments. ** P < 0.01 vs. control group, ##P < 0.01 vs. VIP group.
cultures treated with 108 M VIP after a 24 h of incubation (SPF: 30.8 1.9% (control) versus 38.3 1.2% (VIP), P < 0.05; G2/M-phase fraction: 16.4 0.5% (control) versus 18.6 1.1% (VIP), P < 0.05). Addition of H-7 (SPF = 22.2 2.8, 2.8, PI = 33 3.3), W-7 (SPF = 19.5 3.3, PI = 32.5 3.9), H89 (SPF = 31.9 3.9, PI = 45.8 3.2), and PD98059 (SPF = 28.3 2.5, PI = 36.5 4.3) partially reversed proliferate effects by VIP (Fig. 4).
3.4. Effects of H-7, W-7, H-89, PD98059 on VIP-induced expression of E-cadherin in HBEC We observed the effects of H-7, W-7, H-89, and PD98059 on expression of the E-cadherin in HBEC. These inhibitors alone did not alter the expression of E-cadherin mRNA or protein in
peptides 28 (2007) 1667–1673
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Fig. 6 – Effects of H-7, H-89, W-7, and PD98059 on the expression of E-cadherin mRNA. HBEC cultures were incubated with vehicle (control), H-7, H-89, W-7, and PD98059 (10S5 M for all of these inhibitors) in the presence of VIP (10S8 M) for 20 h. After incubation, total RNA from each treatment was extracted, and the expression of E-cadherin mRNA was measured by RT-PCR. Representative gels from six experiments in each condition are shown. (A) The relative intensity of E-cadherin mRNA (E-cadherin/GAPDH) is shown on the bottom panel. (B) Data are mean W S.E. from five separate experiments. ** P < 0.01 vs. control group, ##P < 0.01 vs. VIP group.
HBEC in the absence of VIP (data not shown). Consistent with our previous findings, VIP (108 M) increased the expression of E-cadherin mRNA and protein after 28 h of incubation in HBEC [6]. However, pretreatment with H-7 (105 M), W-7 (105 M), H-89 (105 M) or PD98059 (105 M) decreased the expression of E-cadherin protein in the presence of VIP (Fig. 5A and B). RTPCR analysis indicated that H-7 (105 M), W-7 (105 M), H-89 (105 M), and PD98059 (105 M) also decreased the expression of E-cadherin mRNA in the presence of VIP (Fig. 6A and B).
4.
Discussion
Bronchial epithelial cells play an important role in maintaining the integrity of the epithelium barrier and the healing of bronchial injuries in various respiratory diseases. The structural defect and dysfunction of airway epithelium are initial processes in airway hyper-responsive diseases. Repairing of bronchial epithelium mainly depends on migration and
proliferation of bronchial epithelial cells. Interactions between cell adhesion molecules, such as E-cadherin, at the cell surface are essential for repair to occur. Our previous findings indicated that VIP enhanced the migration, proliferation and expression of E-cadherin, suggesting a role of VIP in bronchial epithelial repair [6]. However, the underlying mechanisms responsible for VIP-mediated wound healing are unclear. In the present study, we have found that the VIP receptor downstream signaling molecules are involved in these processes. It is well known that VAPC is coupled to the PKA, PKC, CaM, and MAPK transduction pathways. For example, VIP increases the expression of aquaporin-3 via a PKA-dependent pathway in a human colonic epithelial cell [9], and it stimulates glibenclamide-sensitive and DIDS-insensitive iodide efflux through activation of PKA and PKC in human bronchial epithelial Calu-3 cells [3]. Furthermore, VIP-induced migration of rabbit airway epithelium has been shown to be linked to CaM and PKC pathways [7]. However, it is also reported that
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references
Fig. 7 – Proposed mechanisms for VIP-mediated protections in HBEC.
VIP stimulates production of IL-8 in intestinal epithelial cells via a PKA-independent, MAPK-dependent pathway [21]. PKC inhibitor H-7 (105 M) and the calmodulin inhibitor W-7 (105 M) can reverse VIP-mediated effects on LPSinduced MMP-9 activity [16]. PKA inhibitor H-89 reverses the inhibitory effect of VIP (107 M) on LPS-induced TNF-a production at the concentration of 105 M [14]. The blockade of the MAPK cascade with PD98059 (105 M) also completely abolishes VIP-induced ERK1/2 activation in rat anterior pituitary cells [4]. Our present study shows that H-7, W-7, H-89, and PD98059 are able to inhibit VIP-induced migration, proliferation, and the expression of E-cadherin of HBEC. These results suggest that the PKC, CaM, PKA, and MAPKcontrolled pathways have important roles in VIP-mediated wound healing of HBEC. PKC has been shown to regulate recycling of E-cadherin [15], and PKA can affect the cleavage, secretion, and motion of cells via the change of structure and function of microtubule protein by phosphorylating target proteins [20]. CaM also regulates the proliferation, differentiation, and motion by participating DNA synthesis, repair, microtubule depolymerization, and cell cycle-dependent gene expression [22]. Furthermore, MAPK could mediate the damage and apoptosis of the cells [5,23]. Therefore, it seems that these VIP receptor-associated downstream molecules are integrated into the intracellular signal transduction pathways in VIP-mediated biological activities of respiratory epithelial cells. They might act in different phases of wound healing or interact with each other during structural remodeling processes (Fig. 7).
Acknowledgements This work was supported in part by the National Natural Science Foundation of China (No. 39800053). We thank Dr. Gruenert from the University of California, San Francisco, for providing an immortalized human BEC line 16HBE14o-.
[1] Adam EC, Holgate ST, Fildew CJ, Lackie PM. Role of carbohydrates in repair of human respiratory epithelium using an in vitro model. Clin Exp Allergy 2003;33: 1398–404. [2] Corbitt J, Hagerty T, Fernandez E, Morgan WW, Strong R. Transcriptional and post-transcriptional regulation of tyrosine hydroxylase messenger RNA in PC12 cells during persistent stimulation by VIP and PACAP38: differential regulation by protein kinase A and protein kinase Cdependent pathways. Neuropeptides 2002;36:34–45. [3] De´rand R, Montoni A, Bulteau-Pignoux L, Janet T, Moreau B, Muller JM, et al. Activation of VPAC1 receptors by VIP and PACAP-27 in human bronchial epithelial cells induces CFTR-dependent chloride secretion. Br J Pharmacol 2004;141:698–708. [4] Ferna´ndez M, Sa´nchez-Franco F, Palacios N, Sa´nchez I, Cacicedo L. IGF-I and vasoactive intestinal peptide (VIP) regulate cAMP-response element-binding protein (CREB)dependent transcription via the mitogen-activated protein kinase (MAPK) pathway in pituitary cells: requirement of Rap1. J Mol Endocrinol 2005;34:699–712. [5] Goke M, Kanai M, Lynch-Devaney K, Podolsky DK. Rapid mitogen-activated protein kinase activation by transforming growth factor alpha in wounded rat intestinal epithelial cells. Gastroenterology 1998;114:697–705. [6] Guan CX, Zhang M, Qin XQ, Cui YR, Luo ZQ, Bai HB, et al. Vasoactive intestinal peptide enhances wound healing and proliferation of human bronchial epithelial cells. Peptides 2006;27:3107–14. [7] Guan CX, Zhang CQ, Qin XQ, Luo ZQ, Zhou FW, Sun XH. Effects of vasoactive intestinal peptide on chemotaxis of bronchial epithelial cells. Acta Physiol Sin 2002;54:103–6. [8] Hodges RR, Zoukhri D, Sergheraert C, Zieske JD, Dartt DA. Identification of vasoactive intestinal peptide receptor subtypes in the lacrimal gland and their signal-transducing components. Invest Ophthalmol Vis Sci 1997;38:610–9. [9] Itoh A, Tsujikawa T, Fujiyama Y, Bamba T. Enhancement of aquaporin-3 by vasoactive intestinal polypeptide in a human colonic epithelial cell line. J Gastroenterol Hepatol 2003;18:203–10. [10] Itri J, Colwell CS. Regulation of inhibitory synaptic transmission by vasoactive intestinal peptide (VIP) in the mouse suprachiasmatic nucleus. J Neurophysiol 2003;90:1589–97. [11] Jiao W, Miyazaki K, Kitajima Y. Exogenous expression of Ecadherin in gallbladder carcinoma cell line G-415 restores its cellular polarity and differentiation. Int J Oncol 2001;19:1099–107. [12] Jiao W, Miyazaki K, Kitajima Y. Inverse correlation between E-cadherin and Snail expression in hepatocellular carcinoma cell lines in vitro and in vivo. Br J Cancer 2002;86:98–101. [13] Kajita H, Omori K, Matsuda H. The chloride channel ClC-2 contributes to the inwardly rectifying Cl-conductance in cultured porcine choroid plexus epithelial cells. J Physiol 2000;523:313–24. [14] Kim WK, Kan YQ, Ganea D, Hart RP, Gozes I, Jonakait GM. Vasoactive intestinal peptide and pituitary adenylyl cyclase-activating polypeptide inhibit tumor necrosis factor-a production in injured spinal cord and in activated microglia via a cAMP-dependent pathway. J Neurol Sci 2000;20:3622–30. [15] Le TL, Joseph SR, Yap AS, Stow JL. Protein kinase C regulates endocytosis and recycling of E-cadherin. Am J Physiol Cell Physiol 2002;283:489–99.
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[16] Liu YP, Guan CX, Bai HB. Effects of vasoactive intestinal peptide on LPS-induced MMP-9 expression by alveolar macrophages in rats. Zhong Nan Da Xue Xue Bao Yi Xue Ban 2005;30:645–9. [17] Luo SM, Tan WM, Deng WX, Zhuang SM, Luo JW. Expression of albumin, IGF-1, IGFBP-3 in tumor tissues and adjacent non-tumor tissues of hepatocellular carcinoma patients with cirrhosis. World J Gastroenterol 2005;11:4272–6. [18] Makhlouf GM, Murthy KS. Signal transduction in gastrointestinal smooth muscle. Cell Signal 1997;9:269–76. [19] Mazzocchi G, Malendowicz LK, Neri G, Andreis PG, Ziolkowska A, Gottardo L, et al. Pituitary adenylate cyclaseactivating polypeptide and PACAP receptor expression and function in the rat adrenal gland. Int J Mol Med 2002;9: 233–43.
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[20] Taylor SS, Yang J, Wu J, Haste NM, Radzio-Andzelm E, Anand G. PKA: a portrait of protein kinase dynamics. Biochim Biophys Acta 2004;1697:259–69. [21] Toumi F, Neunlist M, Denis MG, Oreshaova T, Laboisse CL, Galmiche JP, et al. Vasoactive intestinal peptide induces IL-8 production in human colonic epithelial cells via MAP kinase-dependent and PKAindependent pathways. Biochem Biophys Res Commun 2004;317:187–91. [22] Vogel HJ. Calmodulin: a versatile calcium mediator protein. Biochem Cell Biol 1994;72:357–76. [23] Waetzig GH, Seegert D, Rosenstiel P, Nikolaus S, Schreiber S. p38 mitogen-activated protein kinase is activated and linked to TNF-alpha signaling in inflammatory bowel disease. J Immunol 2002;168:5342–51.
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/peptides
Intracellular signaling molecules involved in vasoactive intestinal peptide-mediated wound healing in human bronchial epithelial cells Cha Xiang Guan a,*, Yan Ru Cui a, Min Zhang a, Hong Bo Bai a, Ravitej Khunkhun b, Xiang Fang b a b
Department of Physiology, Central South University Xiangya Medical School, Changsha, Hunan 410078, China Department of Medicine, Harbor Hospital Center, Baltimore, MD 21225, USA
article info
abstract
Article history:
Vasoactive intestinal peptide (VIP), a non-adrenergic, non-cholinergic neuromediator, plays
Received 14 February 2007
an important role in maintaining the bronchial tone of the airway and has anti-inflamma-
Received in revised form
tory properties. Recently, we reported that VIP enhances wound repair in human bronchial
14 July 2007
epithelial cells (HBEC). In the present study, we have identified the intracellular signaling
Accepted 17 July 2007
molecules that are involved in VIP-mediated wound healing in HBEC. The effects of VIP on
Published on line 1 August 2007
wound repair of HBEC were partially blocked by H-7 (a protein kinase C (PKC) inhibitor), W-7 (a calmodulin inhibitor), H-89 (a protein kinase A (PKA) inhibitor), and PD98059 (a specific
Keywords:
extracellular signal-regulated kinase (ERK) inhibitor). VIP-induced chemotactic migration
Vasoactive intestinal peptide
was inhibited in the presence of W-7, H-89, PD98059 or H-7. H-7, W-7, and H-89 were also
Human bronchial epithelial cells
found to decrease VIP-induced expression of Ki67 as well as the proliferation index in HBEC.
Wound healing
Furthermore, H-7, W-7, H-89, and PD98059 inhibited the expression of E-cd protein and
PKA
mRNA induced by VIP. These results suggest that intracellular signaling molecules such as
PKC
PKA, PKC, ERK, and calmodulin play important role in VIP-mediated wound healing of HBEC.
Calmodulin
# 2007 Elsevier Inc. All rights reserved.
ERK
1.
Introduction
Vasoactive intestinal peptide (VIP) is an important neuropeptide in the respiratory airway. VIP has many biological activities such as anti-inflammation, relaxation of airway smooth muscles, and inhibition of airway smooth muscle proliferation. VIP exerts its biological activities through the activation of VIP receptors. So far, three VIP receptors (VPAC1,
VPAC2, and PAC1) have been identified. These receptors share a common molecular architecture, which consists of seven transmembrane domains (7TM), three extracellular loops (EC1, EC2, and EC3), three intracellular loops (IC1, IC2, and IC3), a long amino-terminal extracellular domain, and an intracellular carboxyl terminus. There are four main intracellular signal pathways involved in VIP receptor-mediated intracellular signal transduction: (1) induction of adenylate
* Corresponding author. Tel.: +86 731 8836883; fax: +86 731 8830992. E-mail address: [email protected] (Cha Xiang Guan). Abbreviations: VIP, vasoactive intestinal peptide; HBEC, human bronchial epithelial cells; H-7, 1-(5-isoquinolinesulfonyl)-2-methylpi perazine; H-89, dihydrochloride; W-7, N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamid; SPF, S-phase fraction; PD98059, 2-amino-3methoxyflavon; Ki67, proliferating cell nuclear antigen; PI, proliferation index; GAPDH, glyceraldehyde phosphate dehydrogenase; PKA, protein kinase A; PKC, protein kinase C; MAPK, mitogen activated protein kinase; CaM, calmodulin 0196-9781/$ – see front matter # 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2007.07.027
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peptides 28 (2007) 1667–1673
cyclase, and consequently activation of cAMP-dependent protein kinase A (PKA) [2,8,10,13]; (2) activation of protein kinase C (PKC) [16,19]; (3) activation of calmodulin (CaM) [16,18]; (4) phosphorylation of cAMP-dependent mitogen activated protein kinase (MAPK) [4]. Previously, we reported that VIP accelerates wound healing in HBEC. It increases migration and proliferation of HBEC as well as expression of E-cadherin. These effects are blocked by a VIP receptor antagonist [6]. However, it is not known whether VIP receptor-mediated downstream signaling molecules are involved in its protective effects. In this study, we have determined the role of VIP receptors associated with downstream intracellular signaling molecules in the VIPmediated repair processes.
2.
Materials and methods
2.1.
Materials and cell culture
The HBEC line, 16HBE14o- was kindly provided by Dr. Gruenert from the University of California, San Francisco. DMEM/F12 mediums, H-7, W-7, H-89, PD98059, and VIP were purchased from Sigma Chemical (St. Louis, MO, USA). Trypase was from Amresco Company (Ohio, USA). Polycarbonate chemotaxis membrane was obtained from Neclepore Biotech (NJ, USA). DEPC was from BBI (Halifax, NS, Canada). Reverse transcription kits, Taq DNA polymerase, dNTP were from MBI (Ohio, USA). Trizol was from Invitrogen Life Technologies (California, USA). Primer was from Takara (Dalian, China). A DNA marker was from Beijing DingGuo Biotech (Beijing, China). The monoclonal antibodies against human E-cadherin, Ki67, and the secondary antibody were purchased from Beijing Zhongshan Biotech (Beijing, China). DAB/H2O2 coloring reagent was from Wuhan Boster Biological Technology (Wuhan, China). HBEC were cultured in a mixture medium of DMEM: F12 (1:1) containing 100 units/ml of penicillin, 100 units/ml of streptomycin, and 10% bovine calf serum and maintained at 37 8C in a humidified environment with 5% CO2.
2.2.
Wound healing assay
HBEC were cultured in 12-well plates with DMEM: F12 (1:1), and a small wound area was made in the confluent monolayer by mechanical scraping [1]. The wound area from each cultures was measured every 4 h up to 24 h. A linear regression equation of the remaining wound area to time was obtained. The repair index (RI), which is equal to the absolute value of slope, was used to identify the repair speed of HBEC.
2.3.
HBEC chemotaxis
HBEC chemotaxis was assessed by the blind well chamber technique. The confluent HBEC were treated with 0.05% trypsin at 37 8C for 10 min, and then 2.5% bovine calf serum was added to terminate the digestion. HBEC were then collected, and an aliquot of 200 ml HBEC (1.0 106 cells/ml) was loaded into the upper well of the chamber, while VIP or inhibitor was placed in the bottom chamber. The chambers were incubated for another 6 h. After incubation, the cells on
the top of the filter were removed by scraping. The filters were then fixed and stained with W & G stain. The number of cells remaining in the filters was counted with a microscope [6].
2.4.
Immunohistochemistry of Ki67
The cells were fixed with 95% ethanol and 0.1% Triton-X100. The fixed slides were incubated with 3% H2O2, and blocked with a normal goat serum for 20 min. The slides were then incubated with a mouse anti-human Ki67 monoclonal antibody (1:200) at 4 8C for overnight. The secondary antibody was added to the incubation at 37 8C for 30 min, and S-A/HRP was then added to the incubation at 37 8C for another 30 min. The slides were visualized with DAB/H2O2, counterstained with hematoxylin, and differentiated by chlorhydric acid alcohol. The slides were finally mounted with glycerol. The Ki67positive nuclei were evaluated by microscopy. The Ki67positive nuclei were counted through a minimum of 200 cells per field in five different fields. The Ki67 labeling index (Ki67 LI) is the number (%) of positive cells [17].
2.5.
Determination of cell cycling by flow cytometry
The cells were incubated with VIP in the presence or absence of inhibitors for 24 h, and were fixed in 70% ethanol at 4 8C for another 1 h. The cells were then incubated with RNase (100 mg/ ml, 1 ml) at 37 8C for 30 min. The cells were stained with 0.01% propidium iodide, filtered by grit for 20 min. The distribution of cells in the cell cycle was measured by flow cytometry. Adopted S-phase fraction (SPF) and the proliferation index (PI) were used to determine the proliferation of HBEC [6].
2.6.
Immunohistochemistry of E-cadherin
HBEC were fixed, and incubated with a mouse anti-human Ecadherin monoclonal antibody (1:200). After being mounted with glycerol, the slides were observed via microscope. The buffy granules were considered as E-cadherin product. Pictures were taken at 400 magnification, and background gray scales of those pictures were rectified by an Image-Pro Plus 5.0 image analytical system. The average gray scale was analyzed according to the instructions, and the higher the average gray scale value, the higher the expression of positive granules ratio [6].
2.7.
E-cadherin mRNA
The expression of E-cadherin was measured by RT-PCR, and the GAPDH mRNA expression was chosen as a standard. Total RNA was isolated, and 1 mg RNA was reverse transcribed in a 20 ml reaction buffer containing 4 ml 25 mM MgCl2, 2 ml 10 buffer, 0.5 ml RNasin, 15 U avian myeloblastosis virus, 0.5 mg oligo (dT) primer. The primer sequences were as follows: Ecadherin (502 bp) forward: 50 -TCC CAT CAG CTG CCC AGA AA30 , and reverse: 50 -TGA CTC CTG TGT TCC TGT TA-30 ; GAPDH (240 bp) forward: 50 -TGA TGA CAT CAA GAA GGT GGT GAA G30 , and reverse: 50 -TCC TTG GAG GCC ATG TGG GCC AT-30 [11,12]. PCR reaction conditions were as follows: 95 8C for 5 min, 94 8C for 1 min, 58 8C for 2 min, 72 8C for 3 min for 30
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Fig. 1 – Effects of H-7 (10S5 M), H-89 (10S5 M), W-7 (10S5 M), and PD98059 (10S5 M) on closure of HBEC monolayer wounds induced by VIP (10S8 M). The HBEC monolayer was recorded by video microscopy, and the wound area from each culture was calculated by an Image Pro+ System. The remaining wound area/wound area at 0 h (%) of the groups in different time is shown in the top panel, while the RI is shown in the bottom panel. Data are represented as mean W S.E. from three separate experiments. **P < 0.01 vs. control group, ##P < 0.01 vs. VIP group.
cycles, then 72 8C for 5 min. The products were electrophoresed on a 2% agarose gel. The gel was then visualized by ultraviolet light, and the density of E-cadherin mRNA was measured [6].
Fig. 3 – Effects of H-7 (10S5 M), H-89 (10S5 M), W-7 (10S5 M), and PD98059 (10S5 M) on VIP-induced proliferation of HCEC. (A) The presence of Ki67 from each culture was determined by immunochemical method as described in Section 2. Ki67-positive nuclei were stained brown, while the Ki67-negtive nuclei were stained light blue. HBEC cultures were incubated with H-7, H-89, W-7, or PD98059 in the presence of VIP for 24 h. (B) The expression of Ki67 was expressed as the positive cell-labeling index (LI). Data are expressed as mean W S.E. from five separate experiments. **P < 0.01 vs. control, ##P < 0.01 vs. VIP groups.
2.8.
Statistical analyses
The data were expressed by mean S.E., and the difference between the multiple treatment groups was analyzed by ANOVA. P < 0.05 was considered as statistically significant.
Fig. 2 – Effects of W-7 (10S5 M), H-7 (10S5 M), H-89 (10S5 M), and PD98059 (10S5 M) on VIP-mediated HBEC chemotaxis. HBEC cultures were incubated with W-7, H-7, H-89, and PD98059 in the presence of VIP (10S7 M) for 6 h, and the chemotaxis was measured by the blind well chamber technique. Data values are mean W S.E. obtained from six separate experiments. **P < 0.01 vs. control group, ## P < 0.01 vs. VIP group.
3.
Results
3.1. The effect of H-7, W-7, PD98059, H-89 on VIP-mediated wound healing A small wound area in cultured HBEC was generated in each well with an area of approximately 0.832 0.104 mm2 at the beginning as described previously [6]. Consistent with our
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previous findings, VIP significantly improved wound healing in a time-dependent manner. As indicated by RI, protective effects of VIP against mechanical scraping were partially blocked by pretreatment with H-7 (105 M, a PKC inhibitor), W7 (105 M, a calmodulin inhibitor), H-89 (105 M, a PKA inhibitor), and PD98059 (105 M, a specific ERK inhibitor) (Fig. 1). However, pretreatment with H-7, W-7, H-89 or PD98059 in the absence of VIP had no effect on wound healing of HBEC as compared with control (data not shown).
3.2. Effects of H-7, W-7, H-89, and PD98059 on VIP-induced chemotaxis We next determined the effect of VIP on HBEC migration. VIP (107 M) significantly accelerated the chemotactic migration, and the effects of VIP were decreased by pretreatment with W7, H-7, H-89 or PD98059 (the chemotactic intensities: 21.7 3.8 (VIP 107 M), 9.3 1.8 (VIP + W-7 105 M), 11.5 1.6 (VIP + H-7 105 M), 9.8 1.2 (VIP + H-89 105 M), and 11.8 1.2 (VIP + PD98059 107 M) cells/10 hpf) (Fig. 2).
3.3. Effects of H-7, W-7, H-89, and PD98059 on VIP-mediated proliferation of HBEC We first determined whether H-7, W-7, H-89, and PD98059 alone could affect the proliferation of HBEC using Ki67positive cells as an index for cell proliferation. The positive cell-labeling index (LI) in the control group was 9.1 0.9, and H-7, W-7, and PD 98059 alone did not change the Ki67-positive cell-LI (date not shown). VIP (108 M) increased the Ki67positive cell-LI to 12.4 0.9 after a 24 h of incubation (P < 0.01 versus control). H-7 (105 M), W-7 (105 M), H-89 (105 M), and PD98059 (105 M) could partially block VIP-induced Ki67 expression in HBEC (Fig. 3A and B). Furthermore, flow cytometry analysis of HBEC indicated that SPF and G2/M-phase fraction were increased in the
Fig. 4 – Effects of H-7, H-89, W-7, and PD98059 on VIPinduced changes of cell cycling. The HBEC cultures were incubated with vehicle (control) H-7 (10S5 M), H-89 (10S5 M), W-7 (10S5 M), and PD-98059 (10S5 M) in the presence of VIP (10S8 M). After incubation, the cell cycling was analyzed by flow cytometry. Data are expressed as mean W S.E. from three separate experiments, *P < 0.05 vs. control group, # P < 0.05 vs. VIP group, ##P < 0.05 vs. VIP group.
Fig. 5 – Effects of H-7 (10S5 M), H-89 (10S5 M), W-7 (10S5 M), and PD98059 (10S5 M) on the expression of E-cadherin protein. (A) HBEC cultures were incubated with different treatments for 28 h, plasmalemma-associated E-cadherin was then detected by a specific antibody against to Ecadherin. Monolayer images were recorded, and the average intensity was calculated by an Image Pro+ system. (B) Data are mean W S.E. from six separate experiments. ** P < 0.01 vs. control group, ##P < 0.01 vs. VIP group.
cultures treated with 108 M VIP after a 24 h of incubation (SPF: 30.8 1.9% (control) versus 38.3 1.2% (VIP), P < 0.05; G2/M-phase fraction: 16.4 0.5% (control) versus 18.6 1.1% (VIP), P < 0.05). Addition of H-7 (SPF = 22.2 2.8, 2.8, PI = 33 3.3), W-7 (SPF = 19.5 3.3, PI = 32.5 3.9), H89 (SPF = 31.9 3.9, PI = 45.8 3.2), and PD98059 (SPF = 28.3 2.5, PI = 36.5 4.3) partially reversed proliferate effects by VIP (Fig. 4).
3.4. Effects of H-7, W-7, H-89, PD98059 on VIP-induced expression of E-cadherin in HBEC We observed the effects of H-7, W-7, H-89, and PD98059 on expression of the E-cadherin in HBEC. These inhibitors alone did not alter the expression of E-cadherin mRNA or protein in
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Fig. 6 – Effects of H-7, H-89, W-7, and PD98059 on the expression of E-cadherin mRNA. HBEC cultures were incubated with vehicle (control), H-7, H-89, W-7, and PD98059 (10S5 M for all of these inhibitors) in the presence of VIP (10S8 M) for 20 h. After incubation, total RNA from each treatment was extracted, and the expression of E-cadherin mRNA was measured by RT-PCR. Representative gels from six experiments in each condition are shown. (A) The relative intensity of E-cadherin mRNA (E-cadherin/GAPDH) is shown on the bottom panel. (B) Data are mean W S.E. from five separate experiments. ** P < 0.01 vs. control group, ##P < 0.01 vs. VIP group.
HBEC in the absence of VIP (data not shown). Consistent with our previous findings, VIP (108 M) increased the expression of E-cadherin mRNA and protein after 28 h of incubation in HBEC [6]. However, pretreatment with H-7 (105 M), W-7 (105 M), H-89 (105 M) or PD98059 (105 M) decreased the expression of E-cadherin protein in the presence of VIP (Fig. 5A and B). RTPCR analysis indicated that H-7 (105 M), W-7 (105 M), H-89 (105 M), and PD98059 (105 M) also decreased the expression of E-cadherin mRNA in the presence of VIP (Fig. 6A and B).
4.
Discussion
Bronchial epithelial cells play an important role in maintaining the integrity of the epithelium barrier and the healing of bronchial injuries in various respiratory diseases. The structural defect and dysfunction of airway epithelium are initial processes in airway hyper-responsive diseases. Repairing of bronchial epithelium mainly depends on migration and
proliferation of bronchial epithelial cells. Interactions between cell adhesion molecules, such as E-cadherin, at the cell surface are essential for repair to occur. Our previous findings indicated that VIP enhanced the migration, proliferation and expression of E-cadherin, suggesting a role of VIP in bronchial epithelial repair [6]. However, the underlying mechanisms responsible for VIP-mediated wound healing are unclear. In the present study, we have found that the VIP receptor downstream signaling molecules are involved in these processes. It is well known that VAPC is coupled to the PKA, PKC, CaM, and MAPK transduction pathways. For example, VIP increases the expression of aquaporin-3 via a PKA-dependent pathway in a human colonic epithelial cell [9], and it stimulates glibenclamide-sensitive and DIDS-insensitive iodide efflux through activation of PKA and PKC in human bronchial epithelial Calu-3 cells [3]. Furthermore, VIP-induced migration of rabbit airway epithelium has been shown to be linked to CaM and PKC pathways [7]. However, it is also reported that
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references
Fig. 7 – Proposed mechanisms for VIP-mediated protections in HBEC.
VIP stimulates production of IL-8 in intestinal epithelial cells via a PKA-independent, MAPK-dependent pathway [21]. PKC inhibitor H-7 (105 M) and the calmodulin inhibitor W-7 (105 M) can reverse VIP-mediated effects on LPSinduced MMP-9 activity [16]. PKA inhibitor H-89 reverses the inhibitory effect of VIP (107 M) on LPS-induced TNF-a production at the concentration of 105 M [14]. The blockade of the MAPK cascade with PD98059 (105 M) also completely abolishes VIP-induced ERK1/2 activation in rat anterior pituitary cells [4]. Our present study shows that H-7, W-7, H-89, and PD98059 are able to inhibit VIP-induced migration, proliferation, and the expression of E-cadherin of HBEC. These results suggest that the PKC, CaM, PKA, and MAPKcontrolled pathways have important roles in VIP-mediated wound healing of HBEC. PKC has been shown to regulate recycling of E-cadherin [15], and PKA can affect the cleavage, secretion, and motion of cells via the change of structure and function of microtubule protein by phosphorylating target proteins [20]. CaM also regulates the proliferation, differentiation, and motion by participating DNA synthesis, repair, microtubule depolymerization, and cell cycle-dependent gene expression [22]. Furthermore, MAPK could mediate the damage and apoptosis of the cells [5,23]. Therefore, it seems that these VIP receptor-associated downstream molecules are integrated into the intracellular signal transduction pathways in VIP-mediated biological activities of respiratory epithelial cells. They might act in different phases of wound healing or interact with each other during structural remodeling processes (Fig. 7).
Acknowledgements This work was supported in part by the National Natural Science Foundation of China (No. 39800053). We thank Dr. Gruenert from the University of California, San Francisco, for providing an immortalized human BEC line 16HBE14o-.
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