Lymphocyte α-kinase is a gout-susceptible gene involved in monosodium urate monohydrate-induced inflammatory responses
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- Wang, S., Tu, H., Ko, A.M. et al. J Mol Med (2011) 89: 1241. doi:10.1007/s00109-011-0796-5
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The molecular functions and pathophysiologic role of the lymphocyte α-kinase gene (ALPK1) in gout are unknown. We aimed to examine ALPK1 expression in patients with gout and investigate its role in monosodium urate monohydrate (MSU)-induced inflammatory responses. Microarray data mining was performed with six datasets containing three clinical gout and three volunteer samples. Real-time quantitative polymerase chain reaction (qPCR) assay was used to profile ALPK1 mRNA expression in 62 independent samples. RNA interference for ALPK1 suppression in THP1 cells (human monocytic cell line) was used to scrutinize the functional role of ALPK1 in MSU-mediated inflammatory responses, and ALPK1 expression in MSU-treated THP1 cells was determined by qPCR and Western blot analysis. Cytokine mRNA expression in HEK293 cells after incubation with different concentrations of MSU crystals in the presence or absence of ALPK1 was also detected by qPCR, and ERK1/2, p38, and JNK expressions were investigated by Western blot analysis. ALPK1 mRNA was overexpressed in the clinical gout samples. MSU treatment promoted ALPK1 expression at the mRNA and protein levels. Furthermore, ALPK1 knockdown in THP1 cells resulted in a markedly decreased IL-1β, TNF-α, and IL-8 mRNA expression; plasmid ALPK1 transfection and MSU stimulation synergistically increased the mRNA expression of these cytokines in a concentration-dependent manner. The synergistic effect also led to ERK1/2 activation. ALPK1 is a gout-susceptible gene involved in MSU-induced inflammatory responses. It may contribute to the development of gout by enhancing the inflammatory responses via the mitogen-activated protein kinase pathway.
KeywordsALPK1GoutInflammatory responsesMonosodium urate monohydrateMonocytes
The susceptible genes in hyperuricemia or gout are not entirely clear. Genetic mutations account for only a small fraction of the cases of idiopathic hyperuricemia, as well as rare pathway vulnerabilities, such as deficient hypoxanthine–guanine phosphoribosyltransferase and overactive phosphoribosylpyrophosphate synthetase. Our previous complex segregation analysis  and a genome-wide linkage study on 21 multiplex pedigrees of Taiwanese aborigines with gout  demonstrated a linkage for gout at marker D4S2623 (114 cM) on chromosome 4q25 (logarithm of odds = 4.29). When we focused on this region, we found a novel gene—the lymphocyte α-kinase gene (ALPK1). The molecular functions and pathophysiologic role of ALPK1 in gout are unknown.
Background information on ALPK1 is limited. ALPK1 belongs to a protein kinase family that has no sequence homology to the conventional protein kinases . This new family of protein kinases was named α-kinase on the basis of evidence that these protein kinases phosphorylate amino acids located within α-helices. ALPK1 is implicated in the sorting of proteins in apical transport vesicles or the trans-Golgi network (TGN) in kidney Madin–Darby canine kidney (MDCK) cells, suggesting an essential role in the exocytic transport to the apical plasma membrane in epithelial cells .
Acute gouty arthritis is characterized by the deposition of monosodium urate monohydrate (MSU) crystals in articular and periarticular tissues ; such deposition can be asymptomatic or be associated with the pathogenesis of acute, episodic, self-limiting joint inflammation . Cells encountering MSU crystals express a broad array of inflammatory mediators, including interleukin (IL)-1β, IL-8, and tumor necrosis factor (TNF)-α, all of which have been demonstrated to drive and amplify acute gout . MSU crystals can initiate extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling and transcriptional activation through AP-1 and NF-κB, and induce the release of various inflammatory mediators . Specific blockage of the ERK1/2 pathway drastically reduces upregulation of MSU-mediated chemokine production and activation of nuclear factors .
Several studies have demonstrated the importance of innate immunity in acute gouty inflammation. Because ALPK1 might occur during acute attacks of gout according to our epidemiological study, we hypothesized that ALPK1 amplifies the inflammatory response in gout. Therefore, we investigated ALPK1 expression in patients with gout and its role in the inflammatory responses induced by MSU crystals. Furthermore, because previous reports [8, 9] indicated that MSU crystals activate the transcription of multiple cytokines and chemokines through the mitogen-activated protein kinase (MAPK) pathway, we examined whether ALPK1 modulates MSU-mediated phosphorylation of ERK1/2, p38, and JNK in HEK293 cells.
Materials and methods
Reagents and antibodies
Human embryonic kidney 293 (HEK293) and human monocytic leukemia (THP1) cell lines were obtained from the Bioresource Collection and Research Center (BCRC, Taiwan). Uric acid and Limulus amebocyte cell lysate assay kit were purchased from Sigma-Aldrich (St. Louis, MO, USA). To study MAPK activation, ERK1/2 (Thr202/Tyr204), JNK (Thr183/Tyr185), and p38 (Thr180/Tyr182) phospho-specific and phosphorylation state-independent polyclonal antibodies were purchased from New England Biolabs (Beverly, MA, USA). The anti-ALPK1 polyclonal antibody was obtained from Abgent (San Diego, CA, USA) and the anti-GFP from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Internal control antibodies, β-tubulin and β-actin monoclonal antibodies, were purchased from Chemical (Temecula, CA, USA).
Clinical samples were obtained from the Kaohsiung Medical University Hospital. Clinical rheumatologists confirmed all gout/tophi cases using the American College of Rheumatology criteria [10, 11]. A total of 26 men with gout and 42 control subjects from community clinics were recruited to this study. All control subjects had no diagnosis of gout nor were medicated with hypouricemic agents. This study followed all guidelines by the institutional review board, and the Human Ethics Committee of the hospital approved the study design (KMU-IRB-93063 and KMUH-IRB-960477). Written informed consent was obtained from all participants.
Gene expression and microarray profiling
Isolation of RNA, labels, and hybridization
The National Health Research Institute (NHRI) in Taiwan is the associated agency that performed all experiments. All protocols followed previous reports by the NHRI . Briefly, total RNA of human white blood cells was extracted using RNase Midi kits (Qiagen), and the concentration was determined by NanoDrop 2000 (Wilmington, USA). The quality of total RNA samples was examined by Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA) to detect degraded RNA. RNA samples with RNA integrity numbers of <7 were excluded from this study.
Gene expression microarray profiling
Complementary RNA targets were synthesized, amplified, labeled, and purified using the TargetAmp Nano-G Bioti-aRNA Labeling kit (Epicentre) according to the manufacturer’s instructions. Hybridization of labeled probes to Illumina BeadChips Human HT-12v3 was conducted according to the protocol recommended by Illumina. Each HT-12 chip had 48,804 unique 50-mer oligonucleotide probes with an average of 15-fold feature redundancy. Beadchips were scanned on the Illumina BeadArray 500GX reader and the images processed by the Illumina BeadScan software. The Illumina BeadStudio software was used for preliminary data analysis. A model-based background correction method  was used to correct background noise and to normalize the data before analysis.
Data were analyzed using the Gene spring GX11.0 software (Agilent). Spots producing aberrant measurements due to array artifacts or poor quality were manually or automatically flagged and removed from further analysis. A filter was applied to omit measurements where the fluorescent signal was <100 or >41,336.6015625. Genes that did not meet these criteria in at least one out of the six samples were excluded from further analysis. The fold change of a specific gene expression on gout disease, represented by the ratio of signal intensities of the patient to control for a corresponding probe, was obtained accordingly. For further analysis, we retrieved the log2 ratios of the background-corrected intensities. We only included genes whose expression changed significantly across the dataset. More specifically, the expression levels of a gene in at least three samples had to differ by >2-fold from the mean expression level of control samples to be included in the study.
Preparation of MSU crystals
MSU crystals were prepared by modification of previously described methods [14, 15]. Briefly, we added 1.68 g of uric acid (Sigma-Aldrich) to 500 mL of 0.01 M NaOH heated to 70°C. NaOH/HCl was added to maintain the pH between 7.1 and 7.2. The solution was filtered using a 0.22-μM filter and stored at room temperature. After 7 days, the resulting MSU crystals were washed twice with ethanol and once with acetone and air-dried in a tissue culture hood before use. Crystal sizes were verified by polarizing light microscopy. All MSU crystals were determined to be endotoxin-free by Limulus amebocyte cell lysate assay (Sigma-Aldrich)
THP1 cell culture and ALPK1 depletion
Human monocytic leukemia THP1 cells were cultured in RPMI 1640 containing 2 mM l-glutamine, 4.5 g/L glucose, 10 mM HEPES, 1 mM sodium pyruvate, 0.05 mM 2-mercaptoethanol, 100 U/mL penicillin/streptomycin, and 10% fetal bovine serum. Prior to experiments, 2 × 106 cells were seeded on six-well plates in 2 mL of RPMI 1640 per well. THP1 cells were transfected with ALPK1-siRNA or nonspecific siRNA as a negative control using Lipofetamine™ 2000 transfection reagent and Opti-MEM I medium (Invitrogen, CA, USA) according to the manufacturer’s protocol. The culture medium was aspirated 24 h after transfection and replaced with 1 mL of fresh medium containing 1% fetal bovine serum, with or without MSU crystals (50 or 100 μg/mL) for the indicated times.
HEK293 cell culture and ALPK1 transfection
HEK293 cells were cultured in α-MEM medium containing 100 U/mL penicillin/streptomycin and 10% fetal bovine serum. The ALPK1 plasmid was sub-cloned from pSport-Sfi vector containing ALPK1 cDNA. ALPK1 cDNA was amplified using the primer pair 5′-CGGGATCCATGAATAATCAAAAAGTGGTAGCTGTGC-3′/5′-ACGCGTCGACCTATGTGCATGGTTTCTCCATTG-3′ and ligated into the unique restriction sites, BglII/SalI, of the pEGFP-C1 vector (Clontech, CA, USA). The resulting plasmid denoted pEGFP-ALPK1-C1 was confirmed by sequencing. Prior to experiments, HEK293 cells (1.5 × 106) were seeded on six-well plates in 1 mL of α-MEM medium per well and grown for 24 h. Cells were transfected with pEGFP-ALPK1-C1 or pEGFP-C1 using Lipofetamine™ 2000 transfection reagent and Opti-MEM I medium (Invitrogen) according to the manufacturer’s protocol.
Brightfield and fluorescence images of HEK293 cells were acquired on a Zeiss Axiovert 200 microscope (Zeiss system) using an Evolution VF digital camera and Image-Pro Software (Media Cybernetics, Inc.). The cells were collected for RNA extraction at the indicated times. In the study of MSU crystal application, the culture medium was aspirated 24 h after transfection and replaced with 1 mL of fresh medium containing 1% fetal bovine serum, with or without MSU crystals (0, 150, and 300 μg/mL), for the indicated times.
RNA extraction and real-time quantitative PCR
RNA analysis on peripheral blood leukocytes of 62 subjects (23 gout, 39 controls) was obtained from real-time PCR. Total RNA in human peripheral blood leukocyte cells was extracted using the PAXgene Blood RNA Kit and generated cDNA using TaqMan Reverse Transcription Reagents (Applied Biosystems, Foster City, CA, USA). Pre-designed gene-specific TaqMan probes and primer pairs (assay ID: Hs00228473_m1) were used for ALPK1 mRNA generation. Expression experiments were performed in triplicate and with a control without template included in each plate. Reference housekeeping gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was amplified in the same plate and found to be equivalent in all samples. ALPK1 mRNA was calculated from corresponding threshold cycle (CT) values and normalized to the GAPDH expression in each experiment.
THP1 cells (2 × 106) or HEK293 cells were harvested in the indicated time points. RNA was extracted using REzol™ C & T (PROtech Technologies, Taiwan, Republic of China). Reverse transcription was performed using the high-capacity cDNA reverse transcription kit followed by real-time PCR using the Applied Biosystems 7900HT with SYBR Green Master Mix (Applied Biosystems). The primers used for the real-time PCR were 5′-TGAAAAGTTTAAAAACAATCCACAA-3′/5′-GCAAATGGTGGTCAAACTCC-3′ for ALPK1, 5′-ACGAATCTCCGACCACCACT-3′/5′-CCATGGCCACAACAACTGAC-3′ for IL-1β, 5′-AGACAGCAGAGCACACAAGC-3′/5′-ATGGTTCCTTCCGGTGGT-3′ for IL-8, 5′-CAGCCTCTTCTCCTTCCTGAT-3′/5′-GCCAGAGGGCTGATTAGAGA-3′ for TNF-α, and 5′-CCAACCGCGAGAAGATGA-3′/5′-CCAGAGGCGTACAGGGATAG-3′ for β-actin.
Western blot analysis
At the indicated time points, HEK293 or THP1 cells were lysed with lysis buffer containing 20 mM HEPES, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 2% NP-40, 25% glycerol, and a protease inhibitor cocktail (Sigma-Aldrich). Equal amounts of protein extraction were loaded and separated on 10% sodium dodecyl sulfate polyacrylamide gels. After electrophoresis, the proteins from the gel were transferred to polyvinylidene difluoride (NEN Life Science Products, Boston, MA, USA) membranes. Membranes were blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween 20 and then incubated with the selected primary antibody. β-Tubulin or anti-actin monoclonal antibodies (Chemical) were used to confirm equal protein loading. Horseradish peroxidase-conjugated anti-rabbit or anti-mouse (Amersham, NA, UK) was used as the second antibody, respectively. Bands were visualized by chemiluminescence using enhanced chemiluminescence kit (NEN Life Science Products), exposed to X-ray film for the appropriate time, and quantified by LabWork 4.6 image acquisition and analysis software (UVP, Upland, CA, USA).
Results are expressed as the mean ± SD. The statistical analysis of the data was assessed by using Student’s t test or by a one-way analysis of variance followed by Turkey’s HSD test. Results were considered significant when P > 0.05.
ALPK1 expression in the clinical gout samples
ALPK1 expression after MSU treatment and MSU-induced inflammatory responses upon ALPK1 knockdown
Expression of ALPK1 in HEK293 cells
Synergistic effect of MSU-induced inflammatory responses in ALPK1-transfected HEK293 cells
Regulation of MSU-induced cytokine gene expressions by ALPK1 expression through ERK1/2 and p38 activation
We found that ALPK1 expression is significantly upregulated in patients with gout. In addition, MSU crystals promote ALPK1 expression in THP1 cells at both mRNA and protein levels. The specific interference of AKPK1 RNA reduced the induction of IL-1β, TNF-α, and IL-8 genes by MSU treatment; furthermore, ALPK1 overexpression and MSU treatment synergistically increased the gene expression and production of these cytokines in HEK293 cells along with the induction of ERK1/2 and p38 phosphorylation. In the present study, we demonstrated that rapid induction of ALPK1 by MSU crystals is involved in the MSU-induced inflammatory response that occurs during acute gout attacks. Our results thus provide the initial mechanistic understanding that MSU stimulates ALPK1 expression and protein levels that subsequently affect cytokine expressions, contributing to the overall unpleasant acute gout attack.
Clinical gout is widely known to be an inflammatory arthritis characterized by intra-articular crystallization of MSU . MSU crystals stimulate various types of cells, including monocytes, macrophages, neutrophils, and synovial cells, resulting in the increased production of pro-inflammatory cytokines and chemokines . Several studies have indicated that the release of these inflammatory mediators plays an important role in the infiltration and activation of inflammatory cells in acute gout [7, 14]. In vitro studies indicated that differentiated macrophages are capable of ingesting urate crystals in a non-inflammation manner . Freshly isolated human monocytes show a vigorous response by the induction of TNF-α, IL-1β, IL-6, IL-8, and cyclooxygenase-2 secretion, whereas human macrophages differentiated in vitro for 7 days fail to secrete cytokines or induce endothelial cell activation . However, there is extensive evidence for NLRP3 inflammasome activation in macrophages linking these differentiated cells to triggering inflammation in gout [15, 20, 21]. More recently, in a peritoneal murine model of gout, the resident macrophages, rather than infiltrating monocytes or neutrophils, are important for initiating and driving the early pro-inflammatory phase of acute gout. MSU crystal-recruited monocytes differentiate into a pro-inflammatory M1-like macrophage phenotype in vivo and, combined with ongoing MSU crystal deposition, may play a significant role in abrogating resolution and perpetuating inflammation in gout [22, 23]. These findings suggest that monocyte/macrophage lineage plays a pivotal role not only in the initiation but also in the progression and resolution of acute gouty inflammation. Further studies may be required to assess whether ALPK1 plays a role in the progression.
ALPK1 belongs to a newly discovered protein kinase family that has no sequence homology to the conventional protein kinases, and its catalytic domains appear to be homologous to the catalytic domain of myosin heavy chain kinase A from Dictyostelium . Interestingly, TRPM7/ChaK1 channel kinase, another member of this family, has been shown to phosphorylate annexin 1, a Ca2+- and phospholipid-binding protein that can promote Ca2+-dependent membrane fusion . The pattern of expression of ALPK1 is very similar to that of ChaK1 [25, 26], suggesting that these two proteins have a similar mode of regulation and, perhaps, related functions. Recently, ALPK1 has been shown to function in apical protein transport by phosphorylating myosin 1a , suggesting that an α-kinase is capable of regulating intracellular trafficking processes by phosphorylation. In this study, we found that ALPK1 overexpression and exposure to MSU crystals synergistically increase the phosphorylation of ERK1/2 and p38. Therefore, ALPK1 may act synergistically with MSU crystals in promoting the production of pro-inflammatory cytokines through ERK1/2 and p38 activation. In fact, studies have shown that MSU crystals activate multiple pathways, including phospholipase A2, C and D, Src tyrosine kinases, G protein, and MAPK [8, 27–29]. Further investigation is required to determine the involvement of ALPK1 in the other pathways.
Mechanisms for the synergistic effect of ALPK1 on MSU-induced inflammatory responses have been postulated. ALPK1-mediated phosphorylation of ERK1/2 and p38 might also lead to the activation of transcription complexes, which could have a synergistic effect with NF-κB in promoting the expression of pro-inflammatory genes. It has been demonstrated that MSU crystals potentially activate the NF-κB signaling pathway [8, 9, 27]. These findings suggest that ALPK1 may act synergistically with MSU crystals in promoting the production of pro-inflammatory cytokines through the activation of NF-κB and ERK1/2 and p38 phosphorylation. Moreover, ALPK1 expression in lymphoid organs was significantly decreased by piggyBac(PB) insertion in ALPK1PB/PB mice, leading to thoughts that the immune system may be affected by this mutants . It would be interesting to determine whether ALPK1 is involved in responses to other inflammatory stimulation or whether it is more specific to the MSU/NALRP3 inflammasome-dependent inflammatory pathway.
On the other hand, the human urate anion transporter (URAT1) has been demonstrated to mediate urate handling in the human kidney, which is triggered by uricosuric and anti-uricosuric agents that affect urate excretion . ALPK1 is implicated in the sorting of proteins in apical transport vesicles/TGN in kidney MDCK cells , suggesting an essential role in the exocytic transport to the apical plasma membrane in epithelial cells, possibly the new component of the apical protein sorting by phosphorylating the apical membrane lipid raft vesicle-associated motor protein myosin . Myosin interacts with actin in the brush border membrane ; actin in turn interacts with both myosin and ezrin–radixin–moesin, which interacts with the PDZ domain-containing membrane scaffolding proteins . Interestingly, two of the PDZ domain-containing proteins (PDZK1 and NHERF1) interact with URAT1 [34, 35]. Therefore, ALPK1 may regulate the activity of apical membrane URAT1 by enhancing the process of apical sorting. However, because the function of ALPK1 in the kidney is unknown, further investigations should be conducted to elucidate the biologic interactions between ALPK1 and URAT1 in an MSU-induced hyperuricemia animal model.
In conclusion, we hypothesize that rapid induction of ALPK1 may contribute to the enhancement of MSU-induced acute inflammation through synergistic phosphorylation of ERK1/2 and p38. Further investigation is required to determine how ALPK1 participates in the MSU-induced inflammatory responses and to evaluate the pathologic role of ALPK1 in MSU-induced acute inflammation in vivo using a hyperuricemia animal model. Such studies may define the precise role of ALPK1 in acute gouty arthritis and could also provide a novel therapeutic target for the management of acute gout.
This study was supported by grants from Center of Excellence for Environmental Medicine, Kaohsiung Medical University, Ministry of Education, Taiwan (KMU-EM-98-1-1 and KMU-EM-99-1-1), and the National Science Council, Taiwan (NSC97-2314-B-037-007-MY3 and NSC99-2628-B-037-039-MY3).
The authors have declared that no conflict of interest exists.