Mol Biol Rep (2013) 40:6363–6369 DOI 10.1007/s11033-013-2750-9
Gardiquimod inhibits the expression of calcium-induced differentiation markers in HaCaT cells Bo Jia • Xin Luo • Feng-Wei Cheng Lei Li • Dao-Jun Hu • Fang Wang • Sheng-Quan Zhang
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Received: 25 November 2012 / Accepted: 14 September 2013 / Published online: 22 September 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract Toll-like receptor 7 (TLR7) is an important member in pattern recognition receptors families. TLR7 signal pathway is involved in the physiological process in many type cells, but the impact of TRL7 on differentiation in the human keratinocytes is still unknown. In this study, we investigated the expression of TLR7 in keratinocytes, and the effect of TLR7 agonist gardiquimod on the expression of calcium (Ca2?)-induced keratinocytes differentiation markers in HaCaT cells. Immunohistochemistry and western-blotting analysis showed that TLR7 is expressed in basal keratinocytes of normal skin and in the human keratinocyte cell line HaCaT, but not expressed in the keratinocytes of psoriasis lesions. Pretreatment with gardiquimod could down-regulate Ca2?-induced differentiation marker expression and activate Raf-MEK-ERK and PI3K-AKT signal pathways in HaCaT cells. However, specific inhibitors studies showed that the down-regulation of the differentiation markers expression by gardiquimod was not dependent on the activation of these two pathways. TLR7 may play an important role in the pathogenesis of
Bo Jia and Xin Luo contributed equally to this work.
Electronic supplementary material The online version of this article (doi:10.1007/s11033-013-2750-9) contains supplementary material, which is available to authorized users. B. Jia X. Luo F.-W. Cheng L. Li F. Wang S.-Q. Zhang (&) Department of Biochemistry and Molecular Biology, Anhui Medical University, Hefei, Anhui, China e-mail:
[email protected] D.-J. Hu Department of Clinical Laboratory, Chongming Central Hospital, Shanghai, China
psoriasis through regulating the differentiation of the keratinocytes, and will give a new insight into the psoriasis. Keywords Toll-like receptor 7 Gardiquimod Differentiation HaCaT cells Psoriasis
Introduction Toll-like receptors (TLRs) sense conserved molecules from bacteria, viruses and parasites [1] and are an important family in pattern recognition receptors families, which are involved in driving autoimmune inflammation [2, 3]. Update, ten human TLRs have been characterized [4, 5]. TLR7/8 together with TLR3 constitutes a powerful system to detect the genetic material of RNA viruses [5]. TLR7 are localized to be intracellular and recognize single-stranded RNA or synthetic agonist, such as imiquimod and its derivatives. Gardiquimod, one of imiquimod’s derivatives, is a new imidazoquinoline compound and ten times more active than imiquimod and has been proven to bind specifically to TLR7 [6–8]. The interaction of the TLR7 with its ligands leads to a series signaling events including the activation of signal pathways (MyD88) which consequently lead to the secretion of cytokines and expression of costimulatory molecules [9, 10]. TLR7 agonists can also activate extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase in some cell types, such as dendritic cells [11, 12]. It has been documented that abnormal TLR7 expression is involved in some diseases, including psoriasis and systemic lupus erythematous [13–16]. Psoriasis is a common chronic, autoimmune and hyperproliferative skin disease which is a hallmark of abnormal proliferation and differentiation of keratinocytes [17]. It is will be known, a
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program of differentiation leads to the structural and functional maturation of epidermal keratinocytes. A set of genes that are transcribed only during certain stages of differentiation has been identified. Keratinocytes of the basal layer show a high activity of proliferation and express keratin 5 (K5) and keratin 14 (K14). When the basal keratinocytes differentiated into the spinous layer cells, K5 and K14 are downregulated, but keratin 1 (K1) and keratin 10 (K10) are expressed. As these cells differentiate into granular layer cells, they stop to express K1 and K10, and begin to produce late-differentiation products, such as involucrin [18]. The pathogenesis of psoriasis is involved in the abnormal proliferation and differentiation of keratinocytes. Some studies have documented that the differentiation markers show an abnormal expression and location in the psoriasis lesion compared to the normal epidermis. It has been found that involucrin locates in the upper spinous and granular layers of normal skin, whereas in psoriasis lesion, it is detected in the suprabasal layer with high quantity [19–21]. Recently, the effect of TLR7 on the skin has been studied, the TLR7 agonist imiquimod activates dendritic cells in mice skin and induces lesions resembling psoriasis, when imiquimod is used to treat malignant tumors of the skin [22]. TLR7 is expressed in immune cells such as monocytes, dendritic cells and B cells [23, 24], but not in keratinocytes[25, 26], and at a high concentration, imiquimod, independent of TLR7, stimulates cytokines production by keratinocytes possibly through inhibition of adenylyl cyclase and adenosine receptor signaling [27–31]. However, other studies have found the TLR7 is expressed by the keratinocytes, and gardiquimod can activate the TLR7 signal pathway in KCs [32]. Here, we reported that Gardiquimod could inhibit the expression of Ca2?-induced differentiation markers in HaCaT cells, but this effect gardiquimod is not associated with the Raf-MEK-ERK and PI3K-AKT signal pathways.
Materials and methods Patients and samples Ten Chinese patients (five women and five men) with untreated chronic plaque psoriasis were studied. The clinical diagnosis of all subjects was confirmed by at least two dermatologists. Informed consent was obtained prior to the collection of samples, and the study was approved by Ethical Committees of No. 1 Hospital, Anhui Medical University. Biopsies were taken from the extensor forearm; nonlesional skin was biopsied at a distance of at least 1 cm away from the lesions. Ten samples of normal skin were collected from cosmetic purposes surgery. Five-micrometre skin sections were cut with a cryostat and stored at -80 °C until staining.
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Reagents Gardiquimod was purchased from InvivoGen(san Digo). Antibodies against pERK1/2, pAKT1/2/3, b-Actin, keratin 1, involucrin and keratin 5 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Cell culture HaCaT (human keratinocyte cell line) cells were cultured in M154CF medium (Invitrogen Corporation, CA) containing 4 % fetal bovine serum, 100 U/ml of penicillin, and 100 U/ml of streptomycin. After the cells grew to 50–70 % confluence, it was used to perform the treatment with the gardiquimod. Immunohistochemistry Paraffin-embedded skin tissues were cut into 5 mm sections, deparaffinized, rehydrated, and then stained with rabbit anti-TLR7 (1:50). Briefly, the sections were air dried, fixed in cold alcohol for 10 min and washed in phosphate-buffered saline (PBS) for twice. Nonspecific binding was blocked with normal rabbit serum and then slides were incubated with the rabbit anti-TLR7 antibodies (1:50), followed by secondary biotinylated goat anti-rabbit antibody. Finally, specimens were incubated in diaminobenzidine for 5 min, followed by hematoxylin counterstaining, and mounted in aqueous glycergel. Control sections included substitution of the primary antibody with normal rabbit IgG. The positively stained cells showed brown color; photographs were taken using the Image-Pro Plus 5.1 image operation system. Real-time PCR HaCaT cells were seeded in 24-well plates (density at 1 9 105), when the cells confluent up to 70–80 %, the cells were treated with or without gardiquimod for different time. Then we extracted total cellular RNA using Trizol (Invitrogen, Carlsbad, CA). After quantification, 0.5 lg of total cellular RNA was used to perform reverse transcription with a Takara RT kit (DRR037S, Takara) and OligdT primers. The Real-Time PCR was carried out in a 20 ll reaction system containing 200 nM of primers, 50 9 ROX and 2 9 PCR master mix (DRR039A, Takara). All primers (Supplement Table 1) were purchased from Takara Company (Dalian, China). Glyceraldehyde phosphate dehydrogenase was used as control. Samples were amplified in the Applied Biosystem 7500 Real-Time PCR System (Foster city, CA) for 40 cycles with the following conditions: denaturation at 95 °C for 15 s, annealing and extension at 60 °C for 30 s.
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Western blot
Results
After treatment, HaCaT cells were washed twice with cold PBS and then incubated in RIPA buffer containing 1 mM PMSF for 20 min at 4 °C. After quantitative analysis of the protein content by the BCA method, an equal amount of total proteins from each sample were resolved on 12 % SDS-polyacrylamide gels. After electrophoresis, the separated proteins were transferred to polyvinylidene difluoride membrane (Millipore, USA) by electroblotting. The membrane was blocked with Tris-buffered saline(TBS) containing 5 % fat-free milk for 2 h at room temperature, and then incubated in primary antibody/TBS solution overnight at 4 °C. After washing with TBS, the membrane was incubated in HRP-linked secondary antibody/TBS solution for 2 h at room temperature. The bound secondary antibodies were detected with chemiluminescence substrate (Pierce, Woburn, MA) and visualized by radiograph.
TLR7 is expressed in keratinocytes of the normal skin, but not in psoriasis lesion
Statistical analysis
Gardiquimod down-regulated Ca2?-induced Keratin 1 and involucrin expression in HaCaT cells
All experiments were repeated at least three times and representative results are presented. The SPSS13.0 software was use for all statistical analyses. p \ 0.05 was considered to be statistical significance.
The immunohistochemistrical analysis results showed the TLR7 was mainly expressed in the cytoplasm of basal keratinocytes and with weaker staining in suprabasal layers of the nonlesional skin and normal skin (Fig. 1a, c). In contrast, expression of TLR7 was not observed in the lesional psoriatic skin samples (Fig. 1b). The expression of TLR7 in HaCaT cells We extracted the total RNA and protein of HaCaT cells, and then detected the TLR7 protein by reverse transcription PCR and the western-blot. As the results in Fig. 2, the TLR7 mRNA is detected in HaCaT cells (Fig. 2a). The western blot results also confirmed the TLR7 protein is expressed in HaCaT cells (Fig. 2b).
To determine the effect of gardiquimod on Ca2?-induced expression of the differentiation markers Keratin 1 (K1), Keratin 5 (K5) and involucrin (INV) protein in HaCaT cells,
Fig. 1 TLR7 is expressed in basal keratinocytes from normal skin (n = 10). a Nolesional skin from patient with psoriasis; b lesion from psoriasis; c normal skin from heath individual; d lesion from psoriasis as negative control (without TLR7 antibody)
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Fig. 2 TLR7 is expressed in HaCaT cells. a RT-PCR was used to detect TLR7 mRNA level in HaCaT cells and PBMC (peripheral blood mononuclear cell,positive control) from normal individual. b Total cellular proteins were extracted and used to determine TLR7 by Western blot as described in ‘‘Materials and methods’’. Results shown are representative of three separate experiments
the real-time PCR and western blot was performed. Realtime PCR results showed that Ca2? (1 mM) could statistical significantly induce K1 and INV expression, and slightly inhibited the K5 expression although without statistically significant difference (Fig. 3a). After treatment with gardiquimod, the Ca2?-induced K1 and INV expression reduced. In order to investigate whether there was a consistent change in the protein level with the mRNA level, we performed the western-blot assay. The results in Fig. 3b showed that gardiquimod could also down-regulate the Ca2?-induced K1 and INV expression in protein level. This suggested that the gardiquimod could antagonize the Ca2?-induced differentiation of keratinocyte cell line (HaCaT).
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other cell lines [11, 12], and these two pathways is involved in the differentiation of the epidermis [28–30]. We detected the p-ERK1/2 and p-AKT signal pathways in HaCaT cells treated with gardiquimod. The western-blot results (Fig. 4a) indicated that gardiquimod (2 lg/ml) could activate p-ERK1/2 and p-AKT in HaCaT cells. We also analyzed the effect of the specific inhibitor (PD98059 for p-ERK1/2 and LY2940002 for AKT) on blocking this two signal pathways, and the PD98059 and LY2940002 were able to specifically inhibit the phosphorylation of ERK1/2 and AKT respectively (Fig. 4b, c). The effect of gardiquimod repressed the expression of differentiation markers, Keratin 1 and involucrin, was not depend on ERK1/2 and AKT pathway
It has been reported the gardiquimod can activate p-ERK1/2 and p-AKT signal pathways through TLR7 in
As shown in Fig. 4, gardiquimod could activate ERK1/2 and AKT in HaCaT cells. In order to study the relationship between inductive effect of gardiquimod on ERK1/2, AKT activity and expression of Ca2?-induced K1 and INV expression, we co-treated the HaCaT cells with gardiquimod (2 lg/ml) and D098059 (70 lM), LY294002 (32 lM). And then we analyzed K1, K5 and INV expression by realtime PCR and Western blot. Results in Fig. 5 indicated that the gardiquimod could repress the Ca2?-induced K1 and INV expression in mRNA and protein levels (Fig. 5). Whether the Ca2? and/or gardiquimod were present or not, both PD098059 and LY294002 could inhibit the K1 and INV expression (Fig. 5). But the expression of K5 was not significantly changed. The PI3K inhibitor, LY294002, down-regulated these two genes expression far more strongly than the MEK/ERK inhibitor PD098059, while PD098059 only slightly inhibited their expression (Fig. 5).
Fig. 3 Regulation of differentiation markers expression in HaCaT cells. a HaCaT cells were respectively treated with G (Gardiquimod) (2 lg/ml) for 2 h, and then the added Ca (Calcium)(1 mM) into the media and continue to culture for 24 h. The expression of K1(Keratin 1), K5(Keratin 5) and INV(involucrin) was determined by real-time
PCR. The realtime PCR results correspond to the mean and SD from three separate experiments(*p \ 0.05, vs other groups; #p \ 0.05, vs group treated by Ca; #p \ 0.01, vs group treated by Ca); b Western blot analyzed the differentiation markers expression in HaCaT cells above. The results are representative of three separate experiments
Gardiquimod induced the activation of p-ERK1/2, p-AKT in HaCaT cells
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Fig. 4 Gardiquimod-induced phosphorylation of ERK1/2 and AKT, is blocked by specific inhibitors. HaCaT cells were treated with G (Gardiquimod) (2 lg/ml) for the indicated times. Total cellular proteins were extracted and used to determine the p-ERK1/2 and p-AKT by Western blot (a). The cells were pretreated with
PD(PD98059)(70 lM), and LY(LY294002) (32 lM) for 1 h; cells were then treated with gardiquimod (2 lg/ml) for 20 min. Total cellular proteins were extracted and used to determine the phosphorylated and total ERK1/2 (b) and AKT (c). Results are representative of three separate experiments
Fig. 5 Regulation of Keratin 1 and involucrin protein expression by gardiquimod and Ca2? in HaCaT cells. The cells were pretreated with PD98059(70 lM), and LY294002 (32 lM) for 1 h; then gardiquimod (2 lg/ml) was added, and 2 h later, Ca2 ? (1 mM) was added and continue to culture for 24 h. The expression of K1(Keratin 1), K5(Keratin 5) and INV (involucrin) was determined by real-time
PCR(a *p \ 0.05, vs other groups; ##p \ 0.05, vs group treated by Ca; #p \ 0.01, vs group treated by Ca) and Western blot (b). The realtime PCR results correspond to the mean and SD from three separate experiments, and the western blot results are representative of three separate experiments
These results suggested that the effect of gardiquimod repressed the expression of differentiation markers, Keratin 1 and involucrin, was not depend on ERK1/2 and AKT pathway.
Recently, it has been reported that imiquimod, TLR7 ligand, independent of TLR7, stimulates cytokines production by keratinocytes possibly through inhibition of adenylyl cyclase and adenosine receptor signaling, and this effect need a ten times higher concentration of the imiquimod than the one needed to activate TLR7 signal pathway [27–31]. However, other studies have found the TLR7 is expressed by the keratinocytes, and gardiquimod can activate the TLR7 signal pathway in KCs [32]. In our current studies, immunochemistry analysis showed that TLR7 was expressed in basal keratinocytes of the normal skin, but not in the psoriasis lesion. These results suggested that TLR7 is expressed by normal KCs and may be involved in pathogenesis of psoriasis. Calcium triggers activation of multiple pathways to induce differentiation in keratinocytes [33–35]. Some studies have demonstrated that the ERK pathway and PI3K pathway are involved the cell-cycle arrest and differentiation of normal human keratinocytes. However, knockout of
Discussion TLR7 play an important role in immune system and its abnormal expression is involved in some autoimmune disease. In this study, our results demonstrated that the TLR7 was weakly expressed in basal keratinocytes of the normal skin and HaCaT cells, but not in the psoriasis lesion. We found that the activation of TLR7 signal pathway antagonized the effect of Ca2?-induced keratinocytes differentiation in HaCaT cells. Although the gardiquimod, one of TLR7 agonists, could activate the ERK and AKT pathways, the effect of down-regulation of K1 and INV expression by gardiquimod do not depend on these two pathways.
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B-Raf, Raf-1, MEK1, MEK2, ERK1 and ERK2 has no effect on epidermis development and/or homeostasis [36– 38]. Thus, activation of the pathway may be kept under tight control to provide just the right balance of proliferation/ differentiation signals required for epidermal homeostasis [38]. In our study, The MEK inhibitor, PD98059 and the PI3K inhibitor, LY2940002, can repress Ca2?-induced upregulation of the differentiation marker INV. This seems to suggest that the ERK pathway and PI3K pathway can promote the Ca2?-induced expression of differentiation markers. Furthermore, gardiquimod could activate the ERK and AKT pathways, and inhibit the expression of differentiation markers. These results suggested that gardiquimod antagonized Ca2?-induced expression of the differentiation markers is not depend on the activation of these two signal pathways. Some studies have documented that gardiquimod can also activate NF-jB, which is involved in modulating the differentiation of the keratinocytes [39]. Thus, the MEK/ERK and PI3K/AKT pathways may not be a sole target of gardiquimod, other pathways may be involved in the effect of gardiquimod on keratinocytes differentiation. The further studies are required to explore the mechanism of the keratinocytes differentiation regulation by gardiquimod. In addition, whether gardiquimod can activate adenylyl cyclase and adenosine receptor signaling should not been completely excluded in this study, further studies should been performed to explore the effect of gardiquimod on adenylyl cyclase and adenosine receptor signaling pathway. Taking together, we demonstrated that TLR7 was expressed in normal basal keratinocytes, but loss in psoriasis lesion. The inhibitory effect of gardiquimod on the expression of Ca2?-induced differentiation markers is not seemly associated with the activation of Raf-MEK-ERK and PI3K-AKT signal pathways in HaCaT cells. Acknowledgments This work was supported by Grants from the General Program of National Natural Science Foundation of China (81271748), and the Foundation for Doctors, Anhui Medical University. Conflict of interest The authors declared no competing financial interests.
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