Calcitriol inhibits keratinocyte proliferation by upregulating leukocyte elastase inhibitor (serpin B1)

Authors


Abstract

Calcitriol had been proved to be effective for treatment of psoriasis vulgaris. However, the molecular events leading to the normalization of keratinocyte differentiation had not been fully explored. The aim of the study was to evaluate the role of calcitriol and serpin B1 in human keratinocyte cell line (HaCaT) proliferation. Proteins extracted from calcitriol-treated and untreated HaCaT were separated by 2-D differential gel electrophoresis (2DE). Then, the 2DE profiles were analyzed to screen for differentially expressed proteins, which were identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry. The upregulation of serpin B1 was confirmed by quantitative reverse transcription polymerase chain reaction (qRT–PCR) and western blot analysis. The effect of serpin B1 on HaCaT proliferation was analyzed by RNA interference experiments, methylthiazoletetrazolium assay and flow cytometry. Reproducible 2-DE profiles of HaCaT were established. The result that serpin B1 was upregulated in the calcitriol-treated group was confirmed by qRT–PCR and western blot analysis. Calcitriol could inhibit proliferation of HaCaT at the concentration of 10−9–10−6 mol/L. HaCaT proliferation was promoted when serpin B1 was interfered. The inhibition effect of calcitriol was stopped after serpin B1 was interfered. Serpin B1 was overexpressed in calcitriol-treated HaCaT cells and may play an important role in inhibiting HaCaT proliferation by calcitriol.

Introduction

Psoriasis, which affects 2–3% of the population, is a common, chronic and recurrent inflammatory skin disease with pathological manifestations of excessive keratinocyte proliferation and intraepidermal neutrophil accumulations.[1, 2] Calcitriol and its analogs have been proved to be effective for treatment of mild-to-moderate psoriasis,[3, 4] and the mechanism is that calcitriol inhibits keratinocyte proliferation and stimulates keratinocyte differentiation.[5]

Neutrophils often rise in active lesions and peripheral blood of psoriatic patients and produce oxidative stress which has been linked to severity of psoriasis.[2, 6] The neutrophil granule proteases were found to be not only directly responsible for the killing of phagocytosed pathogens, but an excessive release of neutrophil proteases induced tissue damage and impaired the clearance of apoptotic cells in chronic inflammatory diseases.[7-9] The protease regulation played an important role in epidermal barrier function and inflammatory skin diseases.[10] As one of the protease family, neutrophil elastase (NE) is a major secretor product from activated neutrophils and plays an important role in tissue destruction and pathogenesis of psoriasis. NE is overexpressed in the lesions and serum of psoriasis and plays an important role in the pathogenesis of psoriasis.[11] The level of NE was related to the activity of psoriasis and was suggested to be a good marker for diagnosis and follow-up of the activity.[12] Furthermore, a previous study reported that NE was expressed highly in psoriatic lesions and could stimulate proliferation of keratinocytes.[13] Furthermore, it was reported that NE promoted proliferation of the HaCaT cell line and transwell psoriatic organ culture model.[14] Moreover, the imbalance between NE and endogenous protease inhibitors was widely recognized to be one of the causes of psoriasis. Some inhibitors for NE, including skin-derived anti-leukoproteinase[15] and α1-proteinase inhibitor,[16] were all proved to be related to psoriasis. A proteolysis imbalance was implicated in the development of psoriasis.[2]

The serine protease inhibitors (serpins) are a family of proteins characterized by a unique tertiary structure and employing a suicide substrate-like mechanism to neutralize their target proteinases.[17] As a member of the serpin family, serpin B1 is also known as leukocyte elastase inhibitor and a cytoplasm serine protease inhibitor of polymorphonuclear neutrophils.[18] Serpin B1 is a fast-acting stoichiometric inhibitor of NE, proteinase 3 and cathepsin G, which were found in neutrophil granules by a suicide inhibition mechanism.[10] It was reported that serpins were associated with psoriasis and other common inflammatory skin diseases.[13, 19, 20] But the relationship between serpin B1 and psoriasis has not been reported.

The aim of this study was to confirm the differential expression of serpin B1 in calcitriol-treated keratinocytes and its effect on keratinocyte proliferation.

Methods

Cell culture and drug treatment

An immortalized human keratinocyte cell line, HaCaT, was obtained from the Institute of Biochemistry and Cell Biology (Shanghai, China). HaCaT cells were grown in Dulbecco's modified Eagle's medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Biowest, Nuaillé, France) in a humidified atmosphere containing 5% CO2 at 37°C. Calcitriol (Sigma-Aldrich, St Louis, MO, USA) of 10−8 mol/L was added into the culture medium when 80% of cells were fused, with serum-free DMEM as the negative control.

2DE and mass spectrometry

The total proteins of calcitriol-treated and untreated HaCaT were extracted by repeated freezing and thawing, quantified by Bradford assay, and then were separated by 2-D differential gel electrophoresis (2DE). Total protein (100 μg) were run in the first-dimension isoelectric focusing (IEF) with non-linear gradient immobilized pH gradient (IPG) strips (17 cm) of pH 3–10 and a programmed voltage gradient. IPG strips were rehydrated with sample at 50 V and 17°C for 14 h, and then IEF was conducted at 250 V for 30 min, 500 V for 1 h, 1000 V for 1 h, 10 000 V for 5 h, then 10 000 V to reach a total of approximately 60 KVh. After the IEF run was complete, strips were equilibrated with gentle shaking in two steps for 15 min each in equilibration buffer I (2% dithiothreitol [DTT], 0.375 mol/L pH 8.8 Tris–HCl, 6 mol/L urea, 20% glycerol, 2% sodium dodecylsulfate [SDS] bromophenol blue) and equilibration buffer II (DTT was replaced with 2.5% iodacetamide). Afterwards, the IPG strips were conducted in 12% acrylamide gels in the second dimension with 10 mA 50 V for 50 min and then 50 mA 250 V till the bromophenol blue front reached the bottom of the gel. The separated proteins were visualized by silver diamine staining.

The silver-stained 2DE gels were scanned by a UMAX Power Look 2100XL (UMAX Technologies, Dallas, TX, USA) imaging densitometer. Then, the digitized images were analyzed with Image Master 2D Platinum 5.0 software (Amersham Biosciences, Buckinghamshire, UK) to screen differentially expressed proteins by two-sample Student's t-test (P < 0.05) and fold change (>1.1).

Ten of the differentially expressed protein spots were cut from gels and de-stained, dehydrated and digested with trypsin. The resulting digests were identified by the BrukerAutoflex II matrix-assisted laser desorption ionization time-of-flight mass spectrometer (Bruker Daltonics, Bremen, Germany). Spectra were acquired in the 700–3000 m/z range and then processed with Mascot Distiller software version 2.0.0 (www.matrixscience.com). The resulting peak lists were used to identify the corresponding proteins in National Center for Biotechnology Information and Swiss-Prot databases by peptide mass fingerprinting using the Mascot search engine (www.matrixscience.com). Proteins identified with a MOWSE score of more than 66 are reported. The analysis of the digitized images and protein identification by mass spectrometry were completed by the Research Center for Proteome Analysis, Shanghai Institutes for Biological Sciences.

Reverse transcription and quantitative reverse transcription polymerase chain reaction [qRT–PCR]

Total RNA was isolated from prepared HaCaT cells using Trizol (Invitrogen, Carlsbad, CA, USA) reagents and cDNA was synthesized following the manufacturer's protocol (MBI Fermentas, Vilnius, Lithuania). The sequences of the primers for serpin B1 were as follows: forward, 5′-AGGAACAGTTGACTTTGGAA-3′; and reverse, 5′-AAAGAGATCCTGCACACCTA-3′. qRT–PCR was performed using a standard SYBR green PCR kit (TOYOBO, Osaka, Japan), and PCR-specific amplification was applied with an ABI 7300 (Applied Biosystems, Darmstadt, Germany) machine. The relative level of genes (serpin B1, glyceraldehyde 3-phosphate dehydrogenase [GAPDH]) was calculated with the math formula method.

Western blot analysis

The levels of serpin B1 and GAPDH were determined in whole cell extracts from calcitriol-treated HaCaT cells. Protein extracts were separated on an SDS polyacrylamide gel, blotted onto a nitrocellulose membrane (Millipore, Darmstadt, Germany), and incubated with anti-serpin B1 (sc-66957; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-GAPDH (BSAP0063; Bioworld Technology, MN, USA). Immunoblots were developed using goat antirabbit antibody and followed by detection with enhanced chemiluminescence (Pierce, Rockford, IL, USA).

Cell transfection

Three pairs of siRNA options for serpin B1 were designed and synthesized through the Shanghai GenePharma. The sequences were as follows: serpin B1-homo-491 sense, 5′-CGGGCAUGGUUGAUAACAUTT-3′; antisense, 5′-AUGUUAUCAACCAUGCCCGTT- 3′; serpin B1-homo-93 sense, 5′-GGCGUUGAGUGAGAACAAUTT-3′; antisense, 5′-AUUGUUCUCACUCAACGCCTT-3′; serpin B1-homo-813 sense, 5′-GUGGACUAAACCUGAGAAUTT-3′; and antisense, 5′-AUUCUCAGGUUUAGUCCACTT-3′. HaCaT cells were transfected with serpin B1 siRNA for 24 h using Lipofectamine 2000 (Invitrogen, Paisley, UK) according to the manufacturer's instructions. Negative control siRNA was used as a negative control and Lipofectamine 2000 without siRNA used as blank control. Both of the control groups were introduced into the cells using the same protocol. After transfection, cells were cultured in six-well plates at 37°C until needed. The efficiency of gene knockdown was evaluated by qRT–PCR.

Cell proliferation assay by methylthiazoletetrazolium

Three groups of HaCaT cells, which were treated with calcitriol, NE (Calbiochem, Darmstadt, Germany) and DMEM without serum (negative control) separately, were reseeded in 96-well plates at an optimized density 24 h after treatment. Then, 20 μL methylthiazoletetrazolium solution (5 mg/mL) was added into the culture medium for 4 h incubation 24 h later; after which, 150 μL dimethylsulfoxide was added to each well to dissolve the crystals. The absorbance of each sample was recorded at 550 nm after 10 min.

Cell cycle analysis by flow cytometry

HaCaT cells were cultured in six-well plates and then treated with negative control siRNA, blank control, serpin B1 siRNA, negative control + calcitriol (10−8 mol/L) and serpin B1 siRNA + calcitriol (10−8 mol/L), separately. Cells were collected 48 h later, fixed by 70% ethanol for 30 min and then washed with ice-cold phosphate-buffered saline (PBS). The cell pellets were re-suspended in RNase-containing (1:100 in dilution) PBS buffer on ice. At last, the cells were stained with propidium iodide (PI) and then analyzed using a flow cytometer (BD Biosciences, San Jose, CA, USA).

Statistical analysis

The data were shown as means ± standard deviation and were compared by the Student's t-test for unpaired observations. P < 0.05 was considered as significant. The statistical analyses of the data were performed using the SPSS version 18.0 statistical software (SPSS, Chicago, IL, USA).

Results

Serpin B1 was upregulated in calcitriol-treated HaCaT cells

Reproducible 2-DE profiles of HaCaT were established. Protein spots were more than 3000 as shown in Figure 1(a). A total of 22 proteins were differentially expressed in calcitriol-treated HaCaT cells, of which five downregulated and 17 upregulated. Some of them are shown in Figure 1(a) with circles and numbers. Serpin B1, fascin homolog 1, interferon regulatory factor 4 and gi 158256364 were identified to be differentially expressed. Serpin B1 as shown in Figure 1(a) with an arrow was upregulated in the calcitriol-treated group. Figure 1(b–d) shows the process of the identification of serpin B1. In Figure 2(a), as revealed by qRT–PCR analysis, serpin B1 mRNA was upregulated by 1.99, 2.41, 3.28, 3.46 and 3.31 times compared with the control group at 4, 8, 12, 24 and 48 h, separately (P < 0.05). In Figure 2(b), as revealed by western blot analysis, serpin B1 were both upregulated at 24 and 48 h compared with the control group and there was statistical significance (P < 0.05). There was statistical significance between the 24-h group and 48-h group (P < 0.05).

Figure 1.

Serpin B1 identified from proteomic analysis of HaCaT treated with calcitriol (10−8 mol/L). (a) 2-D gel electrophoresis (2DE) of HaCaT cells for proteomic analysis. Differentially expressed proteins were screened out from calcitriol-treated HaCaT cells as shown with circles and numbers. The spot shown with a blue arrow was then identified to be serpin B1. (b) Peptide mass fingerprinting (PMF) of the protein shown with the blue arrow in (a). (c) Probability-based MOWSE score (PBM) of the PMF shown in (b). The red bar with its score out of the shaded region was the result of identification (P < 0.05). (d) Serpin B1 identified as the protein.

Figure 2.

Serpin B1 upregulated after HaCaT was treated with calcitriol (10−8 mol/L). (a) mRNA level of serpin B1 in HaCaT at different time points (4, 8, 12, 24 and 48 h) after 10−8 mol/L calcitriol treatment. (b) Protein level of serpin B1 in HaCaT at 0, 24 and 48 h after 10−8 mol/L calcitriol treatment. *P < 0.05 compared to negative control. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Serpin B1 inhibited HaCaT cell proliferation

Calcitriol inhibited the proliferation of HaCaT at the concentration of 10−9–10−6 mol/L (Fig. 3). In order to determine the role of serpin B1 on proliferation, HaCaT cells were transfected with three serpin B1 siRNA and negative control. The knockdown efficiency of serpin B1 was verified by qRT–PCR. As revealed in Figure 4(a), serpin B1-homo-93 knocked down the expression of serpin B1 effectively by 83%. In Figure 4(b), serpin B1 siRNA promoted HaCaT cell proliferation moderately compared with the negative and blank controls. The result was the same when the serpin B1 siRNA + calcitriol group and negative control + calcitriol group were compared (P < 0.05). The findings indicated that serpin B1 inhibited HaCaT cell proliferation.

Figure 3.

Calcitriol repressed HaCaT cell proliferation. *P < 0.05 compared to negative control. OD, optical density.

Figure 4.

Serpin B1 and calcitriol repressed HaCaT cell proliferation. (a) Identification of serpin B1 knockdown efficiency by siRNA via quantitative reverse transcription polymerase chain reaction. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as fold control. (b) Serpin B1 silencing led to growth promotion in HaCaT cells by 3-(4 5-dimethylthiazol-2-yl)-2 5-diphenyltetrazolium bromide, while calcitriol (10−8 mol/L) inhibited HaCaT proliferation. *P < 0.05 compared to negative control. **P < 0.05 compared to negative control + calcitriol. OD, optical density.

In Figure 4(b), HaCaT proliferation was inhibited in the negative control + calcitriol group compared with the negative control indicating that calcitriol inhibited HaCaT cell proliferation. The result that calcitriol did not inhibit HaCaT proliferation when serpin B1 was knocked down indicated that serpin B1 was essential for calcitriol-mediated proliferation suppression.

In Figure 5(a,c,f), the growth accelerating effect of serpin B1 siRNA was sustained by the data of cell cycle analysis, in which the percentages of G1 phase cells in serpin B1 siRNA and negative control groups were 40.36 ± 3.07% and 47.04 ± 2.15%, respectively (P < 0.05), and the percentages of S phase cells in serpin B1 siRNA and negative control groups were 50.41 ± 4.33% and 35.49 ± 4.30%, respectively (P < 0.05), indicating that serpin B1 siRNA can promote G1/S transition of the cell cycle. As shown in Figure 5(a,e,f), the growth inhibitory effect of calcitriol can also be sustained by the data of cell cycle analysis, in which the percentages of G1 and S phase cells in the negative control + calcitriol group were 61.32 ± 5.59% and 18.85 ± 2.90%, respectively, indicating that a subpopulation of cells were arrested in the G1 phase by calcitriol. There was no significant difference between the negative and blank control groups as shown in Figure 5(a,b,f), and between serpin B1 siRNA and the serpin B1 siRNA + calcitriol group as shown in Figure 5(c,d,f).

Figure 5.

Serpin B1 and calcitriol induced cell arrested in G1 phase. (a–f) Cells with different treatments were analyzed by flow cytometry as described in 'Methods' for cell cycle distribution analysis.

Discussion

Calcitriol and its analog have been proved to be effective for treatment of mild-to-moderate psoriasis. The mechanism was revealed that calcitriol inhibited keratinocyte proliferation and stimulated keratinocyte differentiation.[3, 4] However, the molecular events leading to the normalization of keratinocyte differentiation had not been fully explored. Using a gene expression profiling of 1,25(OH) 2D3-treated keratinocytes, a similar study declared that the expression of serpin B1 was induced in keratinocytes by 1,25(OH) 2D3 treatment. They also demonstrated a vitamin D-regulated differentiation network, which was composed of vitamin D receptor ligands.[21] In the present study, the expressions of several proteins were changed by calcitriol in proteomic analysis. Serpin B1 was focused on and its change was also confirmed. The effect of serpin B1 on HaCaT proliferation was further studied. This study would elucidate the important target protein of calcitriol on HaCaT and provide a new target for psoriasis treatment. This study concluded that serpin B1 was expressed in keratinocytes, and was upregulated in calcitriol-treated HaCaT cells.

Serpin B1 was highly expressed in the cytoplasm of neutrophils.[22] Recently, it had been reported that serpin B1 was also expressed in bronchial and glandular epithelial cells, in addition to neutrophils, macrophages and mast cells.[23] Serpin B1 regulated the innate immune response and reduced tissue damage by the mentioned proteases during inflammation. Serpin B1 was also associated with neutrophil homeostasis in a bacterial infection model in mice.[24-26] A study showed that serpin B1 reduced bacterial counts and inflammatory cell infiltration in a rat model of chronic Pseudomonas aeruginosa infection.[27] In various infection models, it had been shown that serpin B1 was associated with microbial clearance and sustained a healthy neutrophil reserve which is required in immune responses.[28, 29] In conclusion, serpin B1 was known as one of the most efficient inhibitors of NE.[29-31] By inhibiting NE, serpin B1 was proved to play a crucial role in many chronic inflammatory and infectious diseases, such as psoriasis, chronic obstructive pulmonary disease, cystic fibrosis and ulcerative colitis.

In this study, it was also showed that both serpin B1 and calcitriol could inhibit HaCaT cell proliferation. Serpin B1 siRNA could promote G1/S transition of cell cycle while calcitriol could inhibit G1/S transition. If induction of serpin B1 by calcitriol was indeed crucial for proliferation inhibition, knockdown of serpin B1 should rescue the effect of calcitriol on proliferation. To test this, the negative control + calcitriol group and serpin B1 siRNA + calcitriol group were studied. Calcitriol could not inhibit proliferation if serpin B1 was interfered. In conclusion, calcitriol inhibited HaCaT cell proliferation via upregulating serpin B1. Therefore, serpin B1 may serve as the target of calcitriol for psoriasis.

However, further studies are needed to elucidate the effect of serpin B1 on differentiation and apoptosis of HaCaT and the role of serpin B1 in psoriasis in vivo.

In conclusion, this study showed that serpin B1 was expressed in keratinocytes and overexpressed in calcitriol-treated HaCaT cells. Serpin B1 was involved in the treatment of calcitriol for psoriasis vulgaris by inhibiting keratinocyte proliferation.

Author contribution

G. J. and L. H. conceived and designed the experiments; L. H., Z. Y. X. and G. Z. L. performed the experiments; L. H. and Z. Y. X. analyzed the data; and L. H. and Z. Y. X. wrote the paper.

Conflict of interest

All authors in this study declared that there are no conflicts of interest relevant to this manuscript.

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