Proline-Rich Tyrosine Kinase 2 In The Pathogenesis And Treatment Of Systemic Lupus Erythematosus

Backgroud and objectiveProline-rich tyrosine kinase 2 (PYK2)is a non-receptor protein tyrosine kinase that belongs to the focal adhesion kinase(FAK) subfamily. Similar to the prototype FAK,PYK2 contains three structural domains:a centrally located kinase domain, C-terminal domain and N-terminal domain.N-terminal domain contains a receptor combine domain and cytoskeletal protein combine domain.In the C-terminal tail,there are two proline-rich regions that are aa712-713 and aa876-877,PXXPKand PXXLG are domains binding of SH3 domain-containing proteins,including p130cas,Graf, PI3K,Hic-5,paxillin.The centrally located kinase domain contains tree sites of tyrosine phosphorylation402,579,580(Tyr402,Tyr579,Tyr580),which are the active sites of PYK2.Tyr 402 has been shown recently to be the PYK2 autophosphorylation site and was first shown to be bound by the SH2 domain of Src and later to other Src-family protein.Binding of Src is thought to allow for the subsequent phosphorylation of other tyrosine residues on PYK2 leading to increased activity. Phosphorylation of Tyr402 means the activation of PYK2 molecules.PYK2 is primarily expressed in the central nervous system and the hematopoietic system.PYK2 is activated in response to a myriad of stimuli in many different cell types.In response to these stimuli,PYK2 is thought to mediate activation of mitogen-activated protein kinases(MAPK),phosphatidylinositol 3'-kinase(PI3),the Janus kinase/signal transducer and activator of transcription(JAK/STAT) pathway. PYK2 is a signaling molecule that regulates fundamental cellular processes,including adhesion,survival,proliferation,apoptosis differentiation.Because PYK2 is expressed in hematopoietic system,we focus here only on the role of PYK2 in lymphocyte. PYK2 phosphorylation participates to maintain the immunocyte morphological, regulates lymphocytic activity and proliferation,amplifies signals transmitted through various receptors on lymphocyte.Thus,the activation of PYK2 is an important pathway by which a variety of extracellular signals promote lymphocyte activation.Systemic lupus erythematosus is the most representative multi-system involvement in autoimmune disease,it's main feature is the abnormal activation of self-reactive T,B lymphocytes and a variety of the emergence of autoantibodies.The pathogenesis of SLE is still not clear,However,the abnormal activation of lymphocytes resulted by the abnormal regulation of T,B caused by a variety of cytokines,chemokines,growth factors,integrins as well as the outside of physical or chemical irritants (such as ultraviolet light,certain drugs, etc)is an inevitable part of the pathogenesis of SLE,and has become the target goal of treatment of SLE.The present study has confirmed that FAK,members of the same family with PYK2,mediated integrinβ1 signaling promotes SLE patients with T-lymphocyte costimulatory molecules CD40L expression and T lymphocyte activation.Many researches have shown that the abnormal activation of PYK2 and the loss of regulatory response to a number of signaling pathways are the important factors resulted to abnormal cell growth,differentiation, or migration,involved in a variety of tumors,vascular diseases and acute or chronic inflammation,especially those with immune factors involved in the pathophysiological process of inflammation.In view of the important role of PYK2 signaling molecule in the regulation of lymphocyte activation,as well as the important role of the abnormal activation of lymphocytes in the pathogenesis of SLE,We speculate that PYK2 may be involved in the pathogenesis of SLE by interfering with the abnormal activation of lymphocytes. However,the related research about the activation of PYK2 signaling pathway in SLE reported rarely at home and abroad.In this study,we will reseach the activation of PYK2 in SLE by measuring the levels of PYK2 protein and phosphorylation in the peripheral blood mononuclear cells from SLE patients,to explore the function of activated PYK2 by measuring the expression of costimulatory molecules CD40L, CTLA4 protein in SLE patients'PBMCs cultured with promoting or blocking the PYK2 activation.And to explore the role of PYK2 activation in the pathogenesis of SLE.Over half a century ago people began to use corticosteroid to treat systemic lupus erythematosus patients,the hormone therapy has become the mainstream treatment of lupus nephritis. However,high mortality rates of severe lupus nephritis (mainly typeⅣand someⅢ-type) has not been resolved.80s of last century,the U.S. National Institutes of Health (NIH) began to advocate high-dose intravenous therapy of CTX (cyclophosphamide,CTX),and since then,glucocorticoid hormone-binding immunosuppressant drug treatment programs has been a first treatment of choice for SLE.Glucocorticoid and immunosuppressive drugs can inhibite immune response in many aspects,and have important value in SLE patients invovled in important organ damage including lupus nephritis.In the current treatment of SLE,Glucocorticoid and immunosuppressive are the most important drugs.However,in the process of treatment with glucocorticoid and immunosuppressive agents,because of a large quantity of drugs,long duration and prone to side effects or complications, more than 60% of patients died of their toxic side-effects,which seriously affects the patient's survival and quality of life.In recent years,the development of blood purification,biological agents and hematopoietic stem cell transplantation therapy can alleviate the disease to some degrees.But all of these threatments can not achieve the purpose of cure.The treatment of SLE is still a difficult problems to date.Exploring curative effect drugs without side effects to improve the patient's long-term prognosis and quality of life is still clinicians' goal.Curcumin is a component of turmeric,the yellow spice derived from the roots of the plant Curcuma longa used in ancient Asian. Since the first article referring to the use of curcumin to treat human disease was published in The Lancet in 1937,>2,600 research studies using curcumin or turmeric have been published. The mechanisms implicated in the inhibition of tumorigenesis by curcumin are diverse and appear to involve a combination of antiinflammatory,antioxidant,immunomodulatory,pro-apoptotic,and antiangiogenic properties via pleiotropic effects on genes and cell-signaling pathways at multiple levels.Judging from the current animal experiments and clinical application of the effect,curcumin is a new anti-tumor and immuno-modulatory drugs with good curative effect,neither toxic nor side-effects.Curcumin in combination with immunosuppressive agents have good synergy.Curcumin has played an important role in the treatment of cancer,atherosclerosis,infections,multiple sclerosis and rheumatoid arthritis.But for the study about the treatment of SLE with curcumin is currently no reports.In view of a good application prospect of curcumin in the treatment of autoimmune diseases,and the first part of this study has explored that the activation of PYK2 signaling protein may be importantly significant in the pathogenesis and treatment of SLE,to clarify the role of curcumin's anti-autoimmunity is associated with PYK2 or not show a particular importantane.This study will explore the therapeutic effect of curcumin on SLE by determining the effect of curcumin on SLE patients PBMC PYK2 expression and activition.Objects and methodsThe first part of the research studyed the expression and activation and functions of PYK2 signaling protein in SLE PBMCs.48 cases of SLE patients,32 cases of patients with rheumatoid arthritis (RA) and 24 healthy volunteers were selected in the present study.Peripheral blood mononuclear cells(PBMCs) from healthy volunteers, RA patients,and SLE patients were isolated from 20ml heparinized peripheral blood by Ficoll-Paque gradient centrifugation,The isolated PBMCs were divided into two groups:one group was used for Western blotting and Immunocytochemistry;in the other group,the PBMCs were resuspended at 1×109 PBMC/ml in RPMI-1640 medium cultured with PMA or TyrA9.Control cultures without stimulants were included in each experiment.The cultures were incubated at 37℃in a humidified atmosphere containing 5% CO2 for 24h.Conditioned cells were then collected and analyzed for the expression of CD40L and CTLA4 by flow cytometric analysis.PBMCs proliferation was determined with [3H]-thymidine incorporation.The second part of the reseach studyed the impact of curcumin on SLE PBMCs' PYK2 expression and activation.20 cases of SLE patients and 20 healthy volunteers were selected in the present study.PBMCs from healthy volunteers and SLE patients were isolated from 20ml heparinized peripheral blood by Ficoll-Paque gradient centrifugation,The isolated PBMCs were resuspended at 1×106 PBMCs/ml in RPMI-1640 medium cultured with PMA or TyrA9 and PMA or curcumin and PMA.Control cultures without stimulants were included in each experiment.The cultures were incubated at 37℃in a humidified atmosphere containing 5% CO2 for 24h.Conditioned cells were then collected and analyzed for the expression and activation of PYK2 in PBMCs by Western blotting and Immunocytochemistry,the expression of CD40L and CTLA4 mRNA was measured by SYBR green dye I real-time PCR,the expression of CD40L and CTLA4 protein was measured by flow cytometric analysis.PBMCs proliferation was determined with [3H]-thymidine incorporation.Result1,PYK2 is increased and activated in PBMCs from patients with SLE: Quantitative analysis shows PYK2 in PBMCs from SLE,but not RA patients,was significantly up-regulated in inactive and active SLE patients,respectively,compared with that from healthy donors.2,The correlation between the levels of p-PYK2 and clinical manifestation of SLE:the expression of p-PYK2 was markedly up-regulated in PBMCs from SLE patients with class IV lupus nephritis,whereas this up-regulation was not seen in either healthy donors or SLE patients with CNS disease or nephritis other than classⅣ.PYK2 activation showed a significant negative correlation with serum complement level(CH50)(p (0.01,r=-0.668),and a positive correlation with the level of AnuA.PYK2 activation did not show a correlation with the other autoantibodies and the SLEDAI score.3,The effect of PMA on phosphorylation of PYK2 in SLE:Quantitative analysis shows that p-PYK2 in PBMCs stimulated by PMA,but not by medium,was significantly up-regulated in healthy control,RA and SLE patients.4,The impact of phosphorylation of PYK2 in the expression of costimulatory molecules in SLE PBMCs:Using PMA to stimulate PBMCs from active SLE resulted in a significant upregulation of CD40L and CTLA4,whereas this upregulation is not observed in PBMCs pretreated with chemical inhibitor of PYK2 kinase activity (TyrA9).In PBMCs from normal individuals and RA patients,CD40L and CTLA4 expression were also significantly upregulated by stimulation with PMA.This effect, however,cannot be suppressed by administration of TyrA9.5,The phosphorylation of PYK2 promotes the proliferation of SLE PBMCs:The proliferation of PBMCs from all sources were enhanced by PMA.However,in the presence of TyrA9,only PBMCs from SLE patients showed a repressed proliferation when stimulated with PMA.6,Effect of curcumin on the expression and activation of PYK2 in cultured PBMCs from patients with SLE.The expression and activation of PYK2 in normal individuals PBMCs were significantly weaker than those of SLE patient group.Using PMA to stimulate PBMCs,The expression and activation of PYK2 in PBMCs from two groups were upregulated.However, pretreated with curcumin or TyrA9 before PMA stimulus,the expression and activation of PYK2 in PBMs from two groups were decreased. So curcumin can play a role like as tyrosine kinase inhibitor.7,The effect of curcumin on the expression of CD40L,CTLA4 mRNA and protein:PMA induced the upregulation of CD40L,CTLA4 mRNA and protein in PBMCs from SLE as well as normal individuals.Pretreated with curcumin,the expression of CD40L,CTLA mRNA and protein in PBMCs from SLE and normal groups was inhibited.however,TyrA9,PYK2 inhibitor,can only inhibit the expression of CD40L,CTLA4 mRNA and protein in PBMCs from SLE patients,but not from normal groups.8,The effect of curcumin on the proliferation of SLE PBMCs:In vitro, proliferation of PBMCs from normal individuals was weaker than that from SLE patients.With stimulation by PMA,proliferation of PBMCs from SLE was signi-ficantly increased as well as normal idividuals.Pretreated with curcumin,the proliferation of PBMCs from SLE and normal idividuals was inhibited.But,TyrA9 can only inhibit the proliferation of PBMCs from SLE,not from normal individuals.9,The correlation between the effect of curcumin on the activation of PYK2 and the clinical indices in SLE patients:In SLE group,the inhibition rate of PYK2 activation caused by curcumin showed a negative correlation with the level of serum complements (P<0.05) and a positive correlation with quantity of 24 hours' urinary protein(P<0.01,r=0.63).How-ever,we can see that the inhibition rate of PYK2 activation caused by curcumin in SLE patients displayed no correlation with the SLEDAI score.Conclusions and significances1,The expression and activation of PYK2 signaling protein were both elevated in PBMCs from active SLE and inactive SLE patients.which implys that disregulated activation of PYK2 signaling protein may be of pathogenic significance in the abnormal activation of lymphocyte in SLE.2,The expression and activation of PYK2 in SLE PBMCs was significantly higher than that in rheumatoid arthritis,suggesting that the abnormal activation of PYK2 maybe specific to SLE.3,The expression and activation of PYK2 in PBMCs from active SLE and inactive SLE patients were significantly higher,and did not show a correlation with the SLEDAI score,suggesting that disregulated activation of PYK2 signaling protein may be of pathogenic significance in the organ involverrient and progression of SLE in inactive phase.But the mechanism needs further study.4,The expression of p-PYK2 was markedly up-regulated in PBMCs from SLE patients with class IV lupus nephritis,and showed negative correlation with the level of serum complements,positive correlation with the level of AnuA,which imply that PYK2 signaling protein may be associated with the development of Lupus nephritis.5,Using PMA to stimulate PBMCs from active SLE resulted in a significant upregulation of CD40L and CTLA4,whereas this upregulation is not observed in PBMCs pretreated with TyrA9,suggesting PYK2 signaling protein may be a key signaling pathway protein through which PMA-induced the expression of costimulatory molecules CD40L,CTLA4 in SLE PBMCs.6,The proliferation of PBMCs from all sources were enhanced by PMA. However,in the presence of TyrA9,only PBMCs from SLE patients showed a repressed proliferation when stimulated with PMA.Which suggests that the phosphorylation of PYK2 promotes the proliferation of SLE PBMCs.7,The proliferation of PBMCs and expression of costimulatory molecules CD40L and CTLA4 were significantly increased in normal individuals as well as RA patients,but this upregulation was not inhibited by TyrA9,suggesting that besides of PYK2 signaling protein,PMA can also promote the proliferation of PBMCs and the expression of costimulatory molecules CD40L and CTLA4.8,The expression and activation of PYK2 in normal individuals PBMCs were significantly weaker than those of SLE patient group.Using PMA to stimulate PBMCs,The expression and activation of PYK2 in PBMCs from two groups were upregulated.However,pretreated with curcumin or TyrA9 before PMA stimulus,the expression and activation of PYK2 in PBMs from two groups were decreased.So curcumin can play a role like as tyrosine kinase inhibitor.9,PMA induced the upregulation of CD40L,CTLA4 mRNA and protein in PBMCs from SLE as well as normal individuals.Pretreated with curcumin,the expression of CD40L,CTLA mRNA and protein in PBMCs from SLE and normal groups was inhibited.however,TyrA9,PYK2 inhibitor,can only inhibit the expression of CD40L,CTLA4 mRNA and protein in PBMCs from SLE patients,but not from normal groups.Which implys that curcumin may be multi-target inhibitors.10,Curcumin supressed the proliferation of PBMCs through inhibiting the activation of PYK2,So we are promising curcumin may be therapeutic drugs for future interventions in lupus nephritis inflammation.11,In SLE group,the inhibition rate of PYK2 activation caused by curcumin showed a negative correlation with the level of serum complements (P<0.05) and a positive correlation with quantity of 24 hours' urinary protein(P<0.01,r=0.63). Those results suggests that the inhibition of curcumin in patients with SLE was related with patients'condition,especially SLE patients with renal injury.