Clin Rheumatol DOI 10.1007/s10067-014-2599-x
ORIGINAL ARTICLE
Inhibition of transient receptor potential melastatin 7 (TRPM7) channel induces RA FLSs apoptosis through endoplasmic reticulum (ER) stress Xiaofeng Li & Xiaohua Wang & Yarui Wang & Xiaohui Li & Cheng Huang & Jun Li
Received: 23 January 2014 / Revised: 12 March 2014 / Accepted: 29 March 2014 # Clinical Rheumatology 2014
Abstract Transient receptor potential melastatin 7 (TRPM7) is involved in both normal physiological processes and pathology of various diseases. The purpose of this study was to explore the function and underlying mechanisms of TRPM7 channels in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLSs) apoptosis induced by thapsigargin in vitro. In this study, using a combination of Western blotting, RT-PCR, and nuclear morphology analysis, we investigated the influence and potential function of TRPM7 channels on the apoptosis induced by thapsigargin in RA FLSs. Chemical inhibitors (Gd3+ and 2-APB) and specific siRNA for TRPM7 were used to study the role of TRPM7 in RA FLSs apoptosis. The expression of TRPM7 was significantly potentiated in RA FLSs. Co-incubation of RA FLSs with Gd3+, 2-APB, or TRPM7-siRNA increased cell apoptosis. Furthermore, we found that suppression of TRPM7 channels also increased the expression CHOP and calpain and decreased the expression caspase-3. We conclude that suppression of TRPM7 channels may increase RA FLSs apoptosis in vitro, and this is associated with endoplasmic reticulum (ER) stress. Therefore, inhibition of TRPM7 could activate ER stress and induce RA FLSs apoptosis.
Keywords Ca2+ . Endoplasmic reticulum stress . Fibroblast-like synoviocyte . Rheumatoid arthritis . Transient receptor potential melastatin 7 X. Li : X. Wang : Y. Wang : X. Li : C. Huang : J. Li (*) School of Pharmacy, Anhui Medical University, Mei Shan Road, Hefei, Anhui Province 230032, China e-mail: [email protected] J. Li e-mail: [email protected]
Introduction Rheumatoid arthritis (RA) is characterized by tumor-like expansion of the synovium and the subsequent destruction of adjacent articular cartilage and bone [1]. At the same time, angiogenesis is required to maintain the chronic inflammatory state by transporting inflammatory cells to the site of synovitis and supplying nutrients to the pannus [2]. In RA synovium, fibroblast-like synoviocytes (FLSs), the major cell population in invasive pannus [3], actively participate in the inflammatory processes of RA. They produce not only several matrix metalloproteinases but also proinflammatory cytokines such as IL-1 and IL-6 [3] and angiogenic factors such as vascular endothelial growth factor (VEGF) [4]. In addition, RA FLSs proliferate abnormally, show resistance to Fas-mediated apoptosis, and are able to induce cartilage destruction in the absence of other immune cells [5]. In contrast, angiogenesis, the process of new blood vessel formation, is also highly active in RA, particularly during the onset of the disease [6]. Furthermore, newly formed vessels can transport inflammatory cells to synovitis sites and supply nutrients and oxygen to the pannus and thus maintain a chronic inflammatory state [7]. Endoplasmic reticulum (ER) stress is a cellular danger signal, which is triggered by a failure to fold newly synthesized ER proteins. Diverse conditions including hypoxia and low glucose, which are frequently observed in the RA joints, may act as an ER stress to the cells, although evidence of this is lacking. Unless two compensatory mechanisms of unfolded protein response (UPR) and ER-associated degradation work properly, ER stress causes cell damage and eventually cell death [8]. It has become apparent that dysregulated UPR plays an important role in the pathogenesis of some human diseases [9] such as diabetes mellitus [10] and nonalcoholic steatohepatitis [11], in which affected tissues are devoted to extracellular protein synthesis. In addition, C/EBP homologous protein (CHOP, also
Clin Rheumatol
known as GADD153) level is widely used as a sentinel marker of ER stress under pathological conditions [12]. On the other hand, ER stress, implicated in various neurodegenerative processes, increases the level of intracellular Ca2+ and leads to activation of calpain, a Ca2+-dependent cysteine protease [13]. RA shares a common feature with these disease conditions in that synoviocytes and other inflammatory cells produce large amount of proteins like cytochemokines and matrix metalloproteinases that perpetuate arthritic conditions. Transient receptor potential melastatin 7 (TRPM7) channels, members of transient receptor potential channels superfamily, are bifunctional proteins with dual structure of both ion channels and protein kinases [14–16], participating in many important physical processes. Studies have shown that TRPM7 channels are correlated with a variety of diseases such as oxidative stress-induced cell injury [17–19]. Recently, it is suggested that blockade of TRPM7 channels by Gd3+ and 2APB [20, 21] or suppression of TRPM7 expression by siRNA inhibited the proliferation and differentiation of human lung fibroblasts [21]. However, potential functions of TRPM7 channels in the survival of FLSs are still unknown. Thus, we examined the expression of TRPM7 channels in FLSs of rat in the present study. In particular, we assessed the potential correlation of TRPM7 channel expression with FLSs apoptosis induced by ER stress to aim at finding a novel target for the treatment.
Material and methods Materials and reagents 2-Aminoethoxydiphenyl borate (2-APB) and Gd3+ were obtained from Sigma-Aldrich (Sigma-Aldrich, Poole, UK). Dimethyl sulfoxide (DMSO) and MTT (3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenyltetrazoliumbromide) and all other chemicals were purchased from Sigma Inc. (St. Louis, MO, USA). The CHOP, calpain, caspase-3, TRPM7, and β-actin antibodies were purchased from Boster (Wuhan, China), Santa Cruz (CA, USA), Cell Signaling (Beverly, MA, USA), Abcam (Cambridge, UK), and Boster (Wuhan, China), respectively. Secondary antibodies for goat antirabbit IgG HRP and goat-mouse IgG HRP were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). TRPM7, CHOP, and β-actin primers were produced by Shanghai Sangon Biological and Technological Company (Shanghai, China). RA rat model RA rats were induced by 4-week treatment of adult male Sprague–Dawley (SD, 180–200 g) rats with complete Freund’s adjuvant 100 μl per 200 g body weight by paw
injection. Vehicle control rats were injected in the paw with 100 μl of PBS per 200 g body weight at the same time. Synovium in rats was isolated for the further analysis at 28 days after the treatment. SD rats were provided by the Experimental Animal Center of Anhui Medical University. The animal experimental protocol was approved by the University Animal Care and Use Committee. Cell culture FLS were derived from the synovial tissues of RA and vehicle control rats. The cells were cultured in cell culture flasks in high-glucose DMEM medium supplemented with 20 % (v/v) heat-inactivated fetal bovine serum (FBS) (both from Hyclone, USA), 100 U/ml of penicillin, and 100 mg/ml of streptomycin (both from Beyotime, China). Cell cultures were maintained at 37 °C at an atmosphere of 5 % CO2. RT-PCR analysis Total RNA was extracted from tissue and cultured FLS by using TRIzol (Invitrogen) according to the manufacturer’s protocol. The first-strand complementary DNA (cDNA) was synthesized using Thermoscript RT-PCR synthesis kit (Fermentas) according to the manufacturer’s instructions. For reverse transcription-polymerase chain reaction (RT-PCR), the following primer pairs were used: TRPM7 (5′-CTGAAGAGGAATGACTACAC-3′; reverse: 5′-ACAG GAAAAAGAGAGGGAG-3′), CHOP (5′-TCAGATGAAA TTGGG GGCAC-3; reverse: 5′-TTTCCTCGTTGAGCCG CTCG-3′), and β-actin (5′-TGGAATCC TGTGGCATCC ATGAAAC-3′; reverse: 5′-ACGCAGCTCAGTAACAGT CCG-3′). Reverse transcription products were PCRamplified with 35 cycles of 40 sec at 95 °C, 40 sec at 54 °C, and 1 min at 72 °C using ABI9700. The amplified cDNA was run on 1 % agarose gels and visualized under UV light following staining. The band intensities of amplification products were measured by a densitometer, and the results were normalized with β-actin. The results were repeated at least three times independently from three different pools of templates, while each pool of template was extracted from at least eight ventricles. Western blotting analysis Protein isolation and Western blot analysis were performed as previously described [22]. Extracts were separated by 6 or 8 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a polyvinylidene difluoride (PVDF) membrane, which were incubated with primary antibodies against TRPM7 (1:1,000, Abcam, CA, USA), CHOP (1:200, Boster, China), caspase-3 (1:500, Cell Signaling, CA, USA), calpain (1:500, Santa Cruz, CA, USA), and β-actin
Clin Rheumatol
Small interfering RNA silencing FLSs were transfected with 100 nM of small interfering RNA (siRNA) using Lipofectamine 2000 (Invitrogen, CA, USA) according to the manufacturer’s instructions. The oligonucleotide sequences were as follows: TRPM7 siRNA (rat), 5′-GGCACCUUUAUAUCAUUAATT-3′ for the sense strand and 5′-UUAAUGAUA UAAAGGUGCCTT-3′ for the antisense strand. A negative scrambled siRNA (GenePharma, Shanghai, China) was used in parallel. Cells were cultured at 37 °C for 6 h, and then, RT-PCR, Western blotting, and flow cytometer (FCM) were used 72 h after siRNA transfection. Analysis of nuclear morphology Nuclear morphology was detected using the method of Araki et al. [23]. NP-treated cells were fixed with methanol acetic acid for 10 min followed by staining with Hoechst 33342 at
All data are expressed as mean±SEM. Statistical comparisons were made using one-way ANOVA followed by Student’s t test as appropriate. In all cases, p
ORIGINAL ARTICLE
Inhibition of transient receptor potential melastatin 7 (TRPM7) channel induces RA FLSs apoptosis through endoplasmic reticulum (ER) stress Xiaofeng Li & Xiaohua Wang & Yarui Wang & Xiaohui Li & Cheng Huang & Jun Li
Received: 23 January 2014 / Revised: 12 March 2014 / Accepted: 29 March 2014 # Clinical Rheumatology 2014
Abstract Transient receptor potential melastatin 7 (TRPM7) is involved in both normal physiological processes and pathology of various diseases. The purpose of this study was to explore the function and underlying mechanisms of TRPM7 channels in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLSs) apoptosis induced by thapsigargin in vitro. In this study, using a combination of Western blotting, RT-PCR, and nuclear morphology analysis, we investigated the influence and potential function of TRPM7 channels on the apoptosis induced by thapsigargin in RA FLSs. Chemical inhibitors (Gd3+ and 2-APB) and specific siRNA for TRPM7 were used to study the role of TRPM7 in RA FLSs apoptosis. The expression of TRPM7 was significantly potentiated in RA FLSs. Co-incubation of RA FLSs with Gd3+, 2-APB, or TRPM7-siRNA increased cell apoptosis. Furthermore, we found that suppression of TRPM7 channels also increased the expression CHOP and calpain and decreased the expression caspase-3. We conclude that suppression of TRPM7 channels may increase RA FLSs apoptosis in vitro, and this is associated with endoplasmic reticulum (ER) stress. Therefore, inhibition of TRPM7 could activate ER stress and induce RA FLSs apoptosis.
Keywords Ca2+ . Endoplasmic reticulum stress . Fibroblast-like synoviocyte . Rheumatoid arthritis . Transient receptor potential melastatin 7 X. Li : X. Wang : Y. Wang : X. Li : C. Huang : J. Li (*) School of Pharmacy, Anhui Medical University, Mei Shan Road, Hefei, Anhui Province 230032, China e-mail: [email protected] J. Li e-mail: [email protected]
Introduction Rheumatoid arthritis (RA) is characterized by tumor-like expansion of the synovium and the subsequent destruction of adjacent articular cartilage and bone [1]. At the same time, angiogenesis is required to maintain the chronic inflammatory state by transporting inflammatory cells to the site of synovitis and supplying nutrients to the pannus [2]. In RA synovium, fibroblast-like synoviocytes (FLSs), the major cell population in invasive pannus [3], actively participate in the inflammatory processes of RA. They produce not only several matrix metalloproteinases but also proinflammatory cytokines such as IL-1 and IL-6 [3] and angiogenic factors such as vascular endothelial growth factor (VEGF) [4]. In addition, RA FLSs proliferate abnormally, show resistance to Fas-mediated apoptosis, and are able to induce cartilage destruction in the absence of other immune cells [5]. In contrast, angiogenesis, the process of new blood vessel formation, is also highly active in RA, particularly during the onset of the disease [6]. Furthermore, newly formed vessels can transport inflammatory cells to synovitis sites and supply nutrients and oxygen to the pannus and thus maintain a chronic inflammatory state [7]. Endoplasmic reticulum (ER) stress is a cellular danger signal, which is triggered by a failure to fold newly synthesized ER proteins. Diverse conditions including hypoxia and low glucose, which are frequently observed in the RA joints, may act as an ER stress to the cells, although evidence of this is lacking. Unless two compensatory mechanisms of unfolded protein response (UPR) and ER-associated degradation work properly, ER stress causes cell damage and eventually cell death [8]. It has become apparent that dysregulated UPR plays an important role in the pathogenesis of some human diseases [9] such as diabetes mellitus [10] and nonalcoholic steatohepatitis [11], in which affected tissues are devoted to extracellular protein synthesis. In addition, C/EBP homologous protein (CHOP, also
Clin Rheumatol
known as GADD153) level is widely used as a sentinel marker of ER stress under pathological conditions [12]. On the other hand, ER stress, implicated in various neurodegenerative processes, increases the level of intracellular Ca2+ and leads to activation of calpain, a Ca2+-dependent cysteine protease [13]. RA shares a common feature with these disease conditions in that synoviocytes and other inflammatory cells produce large amount of proteins like cytochemokines and matrix metalloproteinases that perpetuate arthritic conditions. Transient receptor potential melastatin 7 (TRPM7) channels, members of transient receptor potential channels superfamily, are bifunctional proteins with dual structure of both ion channels and protein kinases [14–16], participating in many important physical processes. Studies have shown that TRPM7 channels are correlated with a variety of diseases such as oxidative stress-induced cell injury [17–19]. Recently, it is suggested that blockade of TRPM7 channels by Gd3+ and 2APB [20, 21] or suppression of TRPM7 expression by siRNA inhibited the proliferation and differentiation of human lung fibroblasts [21]. However, potential functions of TRPM7 channels in the survival of FLSs are still unknown. Thus, we examined the expression of TRPM7 channels in FLSs of rat in the present study. In particular, we assessed the potential correlation of TRPM7 channel expression with FLSs apoptosis induced by ER stress to aim at finding a novel target for the treatment.
Material and methods Materials and reagents 2-Aminoethoxydiphenyl borate (2-APB) and Gd3+ were obtained from Sigma-Aldrich (Sigma-Aldrich, Poole, UK). Dimethyl sulfoxide (DMSO) and MTT (3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenyltetrazoliumbromide) and all other chemicals were purchased from Sigma Inc. (St. Louis, MO, USA). The CHOP, calpain, caspase-3, TRPM7, and β-actin antibodies were purchased from Boster (Wuhan, China), Santa Cruz (CA, USA), Cell Signaling (Beverly, MA, USA), Abcam (Cambridge, UK), and Boster (Wuhan, China), respectively. Secondary antibodies for goat antirabbit IgG HRP and goat-mouse IgG HRP were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). TRPM7, CHOP, and β-actin primers were produced by Shanghai Sangon Biological and Technological Company (Shanghai, China). RA rat model RA rats were induced by 4-week treatment of adult male Sprague–Dawley (SD, 180–200 g) rats with complete Freund’s adjuvant 100 μl per 200 g body weight by paw
injection. Vehicle control rats were injected in the paw with 100 μl of PBS per 200 g body weight at the same time. Synovium in rats was isolated for the further analysis at 28 days after the treatment. SD rats were provided by the Experimental Animal Center of Anhui Medical University. The animal experimental protocol was approved by the University Animal Care and Use Committee. Cell culture FLS were derived from the synovial tissues of RA and vehicle control rats. The cells were cultured in cell culture flasks in high-glucose DMEM medium supplemented with 20 % (v/v) heat-inactivated fetal bovine serum (FBS) (both from Hyclone, USA), 100 U/ml of penicillin, and 100 mg/ml of streptomycin (both from Beyotime, China). Cell cultures were maintained at 37 °C at an atmosphere of 5 % CO2. RT-PCR analysis Total RNA was extracted from tissue and cultured FLS by using TRIzol (Invitrogen) according to the manufacturer’s protocol. The first-strand complementary DNA (cDNA) was synthesized using Thermoscript RT-PCR synthesis kit (Fermentas) according to the manufacturer’s instructions. For reverse transcription-polymerase chain reaction (RT-PCR), the following primer pairs were used: TRPM7 (5′-CTGAAGAGGAATGACTACAC-3′; reverse: 5′-ACAG GAAAAAGAGAGGGAG-3′), CHOP (5′-TCAGATGAAA TTGGG GGCAC-3; reverse: 5′-TTTCCTCGTTGAGCCG CTCG-3′), and β-actin (5′-TGGAATCC TGTGGCATCC ATGAAAC-3′; reverse: 5′-ACGCAGCTCAGTAACAGT CCG-3′). Reverse transcription products were PCRamplified with 35 cycles of 40 sec at 95 °C, 40 sec at 54 °C, and 1 min at 72 °C using ABI9700. The amplified cDNA was run on 1 % agarose gels and visualized under UV light following staining. The band intensities of amplification products were measured by a densitometer, and the results were normalized with β-actin. The results were repeated at least three times independently from three different pools of templates, while each pool of template was extracted from at least eight ventricles. Western blotting analysis Protein isolation and Western blot analysis were performed as previously described [22]. Extracts were separated by 6 or 8 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a polyvinylidene difluoride (PVDF) membrane, which were incubated with primary antibodies against TRPM7 (1:1,000, Abcam, CA, USA), CHOP (1:200, Boster, China), caspase-3 (1:500, Cell Signaling, CA, USA), calpain (1:500, Santa Cruz, CA, USA), and β-actin
Clin Rheumatol
Small interfering RNA silencing FLSs were transfected with 100 nM of small interfering RNA (siRNA) using Lipofectamine 2000 (Invitrogen, CA, USA) according to the manufacturer’s instructions. The oligonucleotide sequences were as follows: TRPM7 siRNA (rat), 5′-GGCACCUUUAUAUCAUUAATT-3′ for the sense strand and 5′-UUAAUGAUA UAAAGGUGCCTT-3′ for the antisense strand. A negative scrambled siRNA (GenePharma, Shanghai, China) was used in parallel. Cells were cultured at 37 °C for 6 h, and then, RT-PCR, Western blotting, and flow cytometer (FCM) were used 72 h after siRNA transfection. Analysis of nuclear morphology Nuclear morphology was detected using the method of Araki et al. [23]. NP-treated cells were fixed with methanol acetic acid for 10 min followed by staining with Hoechst 33342 at
All data are expressed as mean±SEM. Statistical comparisons were made using one-way ANOVA followed by Student’s t test as appropriate. In all cases, p
