The Mechanism Of Regulator Of Calcineurin1 (RCAN1) Inhibits NF-κB And Its Application In The Lymphoma Treatment

Backgrounds and ObjectiveNuclear transcription factor-KB (NF-κB) has become the hot research point on eukaryotic transcription factors since it was discovered. Researches showed that NF-κB signaling pathways regulate the expression of several kinds of genes which play important roles in significant processes of cells and organism, including cell apoptosis, cellular stress response, innate immunity, thymus development, embryogenesis, inflammatory response, viral infection etc, both in the pathological and physiological processes. Dis-regulation of NF-κB signaling pathways would result in a series of diseases in human and animals, especially chronic inflammation, immune deficiency disorders and tumors.NF-κB is an important transcription factor associated with tumors, and NF-κB was sustained activated in many solid tumors and haematopoietic neoplasms, such as Hodgkin’s lymphoma (HL), acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL). And the protein composition of many viruses can activate NF-κB signaling pathways, ultimately leads to the formation of a tumor. For example, Tax and LMP1, from HTLV-1 and Epstein-Barr virus separately, can activate NF-κB pathways and cause tumor formation. NF-κB signaling pathway was also highly activated in diffuse large B cell lymphoma (DLBCL) infected with EBV. Studies also have found that inhibition of sustained activation of NF-κB can hinder the potential tumorigenicity of the tumorigenic cells, and inhibit the growth of tumor, induce tumor cell apoptosis.The regulator of calcineurin 1 (RCAN1) gene, also known as Down Syndrome Critical Region 1 (DSCR1) gene, plays a critical role in the pathogenesis of Alzheimer’s disease (AD) and Down syndrome (DS). Research found that RCAN1 participates in the dual regulation of calcineurin function, RCAN1 can inhibit calcineurin-NFAT signal pathway via inhibiting the activity of calcineurin, and RCAN1 can be activated by calcineurin-NFAT signal pathway. Moreover, RCAN1 can interact with NIK and NF-κB can promote the transcription of RCAN1.4, regulating the NF-κB signal pathway. All these results showed that RCAN1 plays important roles in the process of biology including cell differentiation, apoptosis and memory. Recent researches showed that disregulation of RCAN1 can lead to several diseases, especially in AD and DS. But it is worth noting that RCAN1 can inhibit tumor angiogenesis via inhibiting calcineurin signal pathway. And up-regulation of RCAN1 will play an extensive anti-tumor effect. In agreement with this, the incidence of several cancers in Down’s syndrome patients is obviously lower than the normal group, such as leukemia and solid tumors.Now, all researches mainly concentrate on that RCAN1 inhibits tumors via inhibiting calcineurin pathway to restrain tumor angiogenesis, and the new mechanism of RCAN1 inhibiting tumor is rare. Whether RCAN1 affect NF-κB and the mechanism of RCAN1 effect on lymphoma needs further research. Our previous study showed that NF-κB can activate the promoter of RCAN 1.4 gene, and promote the RCAN1.4 protein expression. But the effect of RCAN1 on the NF-κB is still unclear. RCAN1 is important in cell differentiation and apoptosis and can inhibit tumor angiogenesis, meanwhile NF-κB is an important transcription factors in tumorigenesis, so we need to find out whether RCAN1 have effect on NF-κB. This topic focuses on the mechanism of RCAN1 affects NF-κB, and try to study the impact of RCAN1 on lymphoma. And on the basic of these previous results, we will discuss the clinical application of RCAN1.Materials and Methods1. Cell culture:HEK293, Raji, CA46, Daudi, Farage were cultured and used for subsequent experiments.2. Detect the regulation of RCAN1 to NF-κB promoter activity:The RCAN1.1 and RCAN1.4 expression plasmids pRCAN1.1-6myc and pRCAN1.4-6myc and the RCAN1 knockdown plasmid si-RCAN1 and their control plasmids were co-transfected with pNF-KBLuc/pRLTK separately into HEK293 cells.48 hours later, the activity of NF-κB promoter was determined during the dual-luciferase gene reporter system. The plasmids pRCAN1.1mychis, pRCAN1.4mychis, si-RCAN1 and the control plasmids was co-transfected with the plasmid pNF-KB into HEK293 cells separately,48 hours after, the cells were harvested and nucleoprotein and cytoplasmic protein were extracted, and the NF-κB levels were determined by WesternBlotting. TBP and β-actin were used as the internal controls respectively. The plasmids si-RCAN 1 and si-con was co-transfected with pRCAN 1.1-6myc into HEK293 cells separately, the protein level of RCAN1 was detected via WesternBlotting to conform the efficiency of si-RCAN 1. β-actin was used as internal control.3. Detect the regulation of RCAN1 to the IκBα protein level:The plasmids pRCAN1.1-6myc, pRCAN1.4-6myc, si-RCAN1 and the control plasmids were transfected into HEK293 cells separately,48 hours after, the total protein of cells was separated by WesternBlotting, and anti-IκBα was used to detect the endogenous IκBα level, anti-myc (9E10) was used to detect the plasmids expression of RCAN1 in HEK293 cells. β-actin was used as the internal controls. The plasmid si-RCAN1 and si-con was co-transfected with pNF-κBLuc/pRLTK separately into HEK293 cells, Bayl 1-7085 and Genistein was added into cells to inhibit IKK and tyrosine kinase separately,48 hours later, the promoter activity of NF-κB was detected via the dual-luciferase gene reporter system. The plasmid pRCAN1.lmychis, si-RCAN1 and the control plasmids was transfected into HEK293 cells separately,48 hours after,200mM H2O2 was added into cells, and the cells were harvested at 0min、 10min、20min、30min、60min, the protein level of endogenous IκBα was determined by WesternBlotting. P-actin was used as the internal control. The plasmids of wide and mutant IκBα, IκBα-myc, IκBα-Y42E-myc and IκBα-Y42F-myc was co-transfected with pRCAN1.lmychis separately into HEK293 cells, and 200mM H2O2 was added into cells 48 hours after transfection, cells were harvested at 0min、 10min、 20min, and the total protein was separated by WesternBlot to detect the exogenous IκBα level. β-actin was used as the internal control. The plasmids pRCAN1.1-6myc, pRCAN1.4-6myc and si-RCAN1 was transfected into HEK293 cells, the antibody anti-IκBα-phospho-Y42 was used in precipitation and anti-IκBα was used in WesternBlotting to detected the effect of RCAN1 on IκBα-Y42 phosphorylation.4. Detect the cell viability of lymphoma cell line Raji、CA46、 Daudi and Farage overexpressed RCAN1:We first constructed the adenoviral vectors carrying RCAN1.1 and RCAN1.4, and purified the recombinant adenovirus via Cesium Chloride density gradient centrifugation. Then obtained titers of infectious purified adenovirus via ultimate dilution method. The recombinant adenovirus Ad-GFP, Ad-RCAN1.1 and Ad-RCAN1.4 were used to infect Raji cells at the MOI 30.48 hours after, the green fluorescence was observed by fluorescence microscopy, then the cells were harvested and RCAN1 expression was tested the in Raji cells, the endogenous IκBα was detected by WesternBlot, the level of caspase3/7 was tested by caspase3/7 detection kit. The cell viability of Raji cells infected recombinant adenovirus Ad-GFP, Ad-RCAN1.1, Ad-RCAN1.1 was detected using CCK8 and lOnM NF-κB activation inhibitor was used as the positive control. The other lymphoma cells CA46. Daudi and Farage were infected with Ad-GFP, Ad-RCAN1.1, Ad-RCAN1.4 at the MOI 60,72 hours later, the green fluorescence was observed by fluorescence microscopy,4-5 days later the cells were harvested and endogenous IκBα was detected by WesternBlot.5. Detected the effect of RCAN1 on SCID mice with lymphoma xenograft tumor: The SCID mice were divided into two groups at random, each group has 10 mice. The Raji cells were infected with recombinant adenovirus Ad-GFP and Ad-RCAN1.1.48 hours later, the cells viability were detected with Trypan Blue, and the viable cell density was adjusted to 1×106～1×107/ml by 1xPBS according to the cell counting. Then the cell suspension was subcutaneous injected into SCID mice,200ul for each mouse. Observe the mice every day, and recorded the tumor diameter and mice weight. The mice were sacrificed and tumors were obtained two weeks after tumorigenesis. The protein level of IκBα in tumors was detected by WesternBlot.6. Detected the protein-protein interaction between RCAN1 and IκBα. and the function area of RCAN1:The plasmid RCAN1.1-6myc was transfected into HEK293 cells, the anti-myc (9E10) antibody was used as precipitation and anti-IκBα was used for the WesternBlot, on the contrary anti-IκBα antibody was used as precipitation and anti-myc (9E10) antibody was used for the WesternBlot. IgG was used as the negative control. So that we detected the interaction of RCAN1 and endogenous IκBα For the interaction between endogenous RCAN1 and endogenous IκBα, we used the RCAN1 N-terminal antibody DCT3 for immunoprecipitation and anti-IκBα for the WesternBlotting. To determine the function area of RCAN1 effect on IκBα, we constructed the deleted mutants plasmids of RCAN1:pRCAN1-1-103. pRCAN1-51-103、pRCAN1-51-172、 pRCAN1-141-172、pRCAN1-141-197, which expresses the 1-103aa,51-103aa, 51-172aa,141-172aa,141-197aa separately. These RCAN1 mutant plasmids and pRCAN1.1-6myc were transfected into HEK293 cells separately, and anti-myc (9E10) antibody was used in precipitation, the anti-IκBα antibody was used in WesternBlot to detect the function area of RCAN1 on IκBα.7. Detect the effect of RCAN1-1-103 on NF-κB and whether RCAN1 impact on NF-κB via calcineurin:The expression plasmids pRCAN1-1-103 and pRCAN1.1-6myc was co-transfected with pNF-KBluc/pRL-TK into HEK293 cells separately.48 hours later, the cells were harvested and the promoter activity of NF-κB was detected by dual-luciferase gene reporter system. We constructed adenovirus vector expressing RCAN1-1-103, Ad-RCAN1-1-103. We used Ad-RCAN1-1-103 to infect the lymphoma cells Raji.4 days later, CCK8 were used to test the cell viability of Raji, and Ad-GFP was used as the control. The plasmids pRCAN1-1-103, pRCAN1.1-6myc, si-RCAN1 and calcineurin expression plasmid was co-transfected with pNFATluc/pRLTK into HEK293 cells separately.48 hours later, the activity of NFAT promoter was determined using the dual-luciferase gene reporter system. The plasmids pRCAN1.1-6myc, si-RCAN1 and calcineurin expression plasmid was co-transfected with pNF-KBluc/pRLTK into HEK293 cells separately.48 hours later, the activity of NF-κB promoter was determined using the dual-luciferase gene reporter system.8. The purification of RCAN1 and RCAN1.1-103 protein and the detection of protein activity:In order to further study the clinical application of RCAN1 inhibiting tumor, we constructed the prokaryotic expression plasmids, Pet28b-RCAN1.1, pet28b-RCAN 1-1-103, pet28b-TAT-RCAN1.1 and pet28b-TAT-RCAN1-1-103. All the plasmid were transformed into BL21(DE3) competence, the positive colonies were picked out to 2ml LB culture solution. After the inducible expression by 0.5mM IPTG and confirmation by Western Blotting, the bacterial cells were cultured to 1L LB overnight. The cells were harvested and pyrolysis using ultrasound. The supernatant after centrifugation was passed through Ni-NTA Resin affinity column. Collected the target protein. Detected the concentration of protein using Bio-Rad DC P Protein Assay and verification the protein using Western Blotting with anti-His antibody. Using the purified protein to culture HEK293 cells, and detected the protein activity by cell immunofluorescence staining.Results1. RCAN1 inhibits the transcriptional activity of NF-κB:The plasmids pRCAN1.1-6myc, pRCAN1.4-6myc, si-RCAN 1 and the control plasmids was co-transfected with pNF-KBLuc/pRLTK separately into HEK293 cells,48 hours after, the activity of NF-κB promoter was obviously inhibited by RCAN1 than the control, while knock-down RCAN1 can significantly increase the promoter activity of NF-κB. The plasmids pRCAN1.lmychis, pRCAN1.4mychis, si-RCAN 1 and the control plasmids was co-transfected with the plasmid pNF-KB into HEK293 cells separately,48 hours after, the Western Blot showed that overexpression of RCAN1 can inhibit nuclear translocation of NF-κB and the cytoplasmic level of NF-κB was increased relatively, while si-RCAN1 can increase the NF-κB nuclear translocation. The effect of si-RCAN 1 knock-down RCAN1 was detected using Western Blotting 48 hours after transfection, and the result showed that si-RCAN1 can reduced the RCAN1 expression obviously.2. RCAN1 inhibits the activity of NF-κB via increasing IIκBα level:IIκBα. is the inhibitor of NF-κB, and prevent its nuclear translocation. The plasmid pRCANl.lmychis, pRCAN1.4mychis, si-RCAN1 and the control plasmids was transfected into HEK293 cells separately, Western Blot showed that RCAN1 overexpressed in HEK293 cells can increase the protein level of IκBα, while knock-down RCAN1 can decrease the IκBα level obviously. At the same time, the 9E10 antibody detection showed that the pRCAN1.lmychis, pRCAN1.4mychis were well expressed in HEK293 cells.3. RCAN1 inhibits the IκBα. phosphorylation at the residue of 42 tyrosine:The plasmids si-RCAN1 and pNF-κBLuc/pRLTK were co-transfected into HEK293 cells, then Bayl 1-7085 and Genistein was added into the cells to inhibit IKK and tyrosine kinase separately, the results showed that use Genistein to inhibit tyrosine kinase decreased the promoter activity of NF-κB, while Bay11-7085 had no effect on NF-κB activity. The 200mM hydrogen peroxide was added to the cells after 48 hours of cell transfection. Western Blot showed that overexpression RCAN1 in HEK293 cells can reduce hydrogen peroxide induced degradation of endogenous IκBα, especially at 10min、20min、30min of cells exposed to H2O2, IκBαlevel was increased obviously, while IκBα level was decreased in a time dependent manner in the control group. Meanwhile knock-down RCAN1 can facilitate hydrogen peroxide induced degradation of endogenous IκBαas well as the control group. The plasmids IκBα-myc, IκBα-Y42E-myc and IκBα-Y42F-myc was co-transfected with pRCAN1.lmychis separately into HEK293 cells,48 hours later, hydrogen peroxide was added to the cells, and total cell protein was extracted, Western Blot showed that in the IκBα, mutant groups, the effect that RCAN1 inhibit hydrogen peroxide induced degradation of exogenous IκBα disappeared, RCAN1 had no effect on IκBα-Y42E or IκBα-Y42F, but RCAN1 still can inhibit wide IκBα degradation. Then the plasmids RCAN1.1-6myc, RCAN1.4-6myc and si-RCAN1 was transfected into HEK293 cells separately, the antibody anti-IκBα-phospho-Y42 was used to precipitation and anti-IκBα was used in Western Blot. The co-IP results showed that RCAN1 overexpression can inhibit the phosphorylation of IκBα-Y42, while knock-down RCAN1 can increase the phosphorylation level of IκBα-Y42.4. RCAN1 reduce the cell viability of lymphoma cell line Raji:The lymphoma cell line Raji was infected by recombinant adenovirus Ad-GFP, Ad-RCAN1.1 and Ad-RCAN1.4 at the MOI is 30.48 hours later, the green fluorescence under the fluorescence microscope indicated that the infection efficiency can reach at 80%-100%, then the cells were harvested, Western Blot showed that the RCAN1 is well expressed in Raji cells, and the protein level of endogenous IκBα:was obviously increased in RCAN1 overexpressed cells, and the caspase3/7 level was also increased than the control. The cell viability of Raji infected by Ad-RCAN1.1 and Ad-RCAN1.4 was decreased obviously than the control tested by CCK8. The other lymphoma cells CA46, Daudi and Farage were infected with Ad-GFP, Ad-RCAN1.1 and Ad-RCAN1.4 at the MOI is 60, the green fluorescence under the fluorescence microscopy was rare, and level of endogenous IκBα had no obviously change.5. Overexpression of RCAN1 suppressed the growth of lymphoma xenograft tumor in SCID mice:Lymphoma cell line Raji infected Ad-GFP、Ad-RCAN1.1 was subcutaneously injected into SCID mice. Lymphoma formation was observed approximately two weeks after Raji cells transplantation. The incidence of xenograft lymphoma was decrease in the RCAN1 overexpressed group at the early stage of tumor formation. And the tumor volume of RCAN1.1 overexpressed group was obviously smaller than the control group. The mice were sacrificed and tumors were dissected. The tumor tissues homogenate were separated by Western Blot, and result showed that the protein level of IκBαwas increased in the RCAN1.1 overexpressed group.6. RCAN1 interacted with IκBα and RCAN1-1-103 is the main functional area:The CO-IP results showed that anti-IκBα detected a band in 9E10 immunoprecipitates that migrated together with IκBαprotein in the cell lysate, and vice versa, the 9E10 antibody detected RCAN1 protein in the anti-IκBα antibody pull-down. So that we make sure that RCAN1 interact with endogenous IκBα. Co-IP also showed that there is a interaction between endogenous RCAN1 and endogenous IκBα:using RCAN1 N-terminal antibody DCT3 for immunoprecipitation and anti-IκBα for Western Blot. To determine the functional area of RCAN1 effect on IκBα, we constructed the deleted mutants plasmids of RCAN1.1:pRCAN1-1-103、 pRCANl-51-103、 pRCAN1-51-172, pRCAN1-141-172、pRCAN1-141-197. The RCAN1 deletion mutant plasmids were transfected into HEK293 cells separately, and anti-myc (9E10) antibody was used in precipitation, the anti-IκBα antibody was used in Western Blot. The Co-IP result showed that only RCAN1-1-103 can interact with endogenous IκBα.So the functional area of RCAN1 effect on IκBα is the N-terminal 1-103.7. RCAN1-1-103 inhibits the activity of NF-κB and the impact of RCAN1 on NF-κB is independent on calcineurin:The expression plasmid pRCAN1-1-103-6myc and pRCAN1.1-6myc, was co-transfected with pNF-KBluc /pRLTK into HEK293 cells separately.48 hours later, dual-luciferase results showed that RCAN1-1-103 can reduce the promoter activity of NF-κB, and its effect is similar to RCAN1.1. We constructed adenovirus vector expressing RCAN1-1-103, Ad-RCAN1-1-103. We used Ad-RCAN1-1-103 to infect the lymphoma cells Raji.4 days later, CCK8 showed that compared to the control, RCAN1-1-103 can reduce cell viability and proliferation of Raji. The plasmids pRCAN1-1-103, pRCAN1.l-6myc, si-RCAN1 and calcineurin expression plasmid was co-transfected with pNFATluc/pRLTK into HEK293 cells separately, 48 hours later, dual-luciferase showed that RCAN1.1 can reduce the promoter activity of NFAT and calcineurin can increase the promoter activity of NFAT, while RCAN1-1-103 had no effect on NFAT. The plasmid pRCAN1.1-6myc, si-RCAN1 and calcineurin expression plasmid was co-transfected with pNF-κBluc/pRLTK into HEK283 cells separately. Dual-luciferase results showed that RCAN1.1 can reduce the promoter activity of NF-κB and calcineurin also inhibit the activity of NF-κB. These results indicated that RCAN1 inhibit the activity of NF-κB is independent on calcineurin.8. The purification of RCAN1.1 and RCAN1-1-103 and the detection of protein activity:We constructed the prokaryotic expression plasmids successfully, pet28b-RCAN1.1, pet28b-RCAN1-1-103, pet28b-TAT-RCAN1.1 and pet28b-TAT-RCAN1-1-103. And we finally purified the protein pet28b-RCAN1.1-103 and pet28b-TAT-RCAN1.1-103. Using the purified protein to culture HEK293 cells, and cell immunofluorescence staining showed that TAT-RCAN1.1-103 can permeate the cell membrane into the cells, while RCAN 1.1-103 can’t permeate the cell membrane.Conclusions1. RCAN1 inhibits the transcription activity of NF-κB and restrain its nuclear localization.2. RCAN1 inhibits NF-κB via increasing the protein level of IκBα3. RCAN1 increases IκBα level by inhibiting Y42 phosphorylation.4. RCAN1 reduces the cell viability of lymphoma cell line Raji via increasing IκBα level and inhibiting NF-κB activity.5. RCAN1 suppressed the growth of xenograft lymphoma in SCID mice via increasing IκBα. level.6. RCAN1 interacts with IκBα and the functional area is RCAN1-1-103.7. The effect of RCAN1 on inhibiting the activity of NF-κB is independent on calcineurin.8. The purified protein TAT-RCAN1.1-103 can permeate the cell membrane into the cells, RCAN 1.1-103 can’t permeate the cell membrane.SignificanceIn conclusion, these findings suggest that RCAN1 inhibits the transcription activity of NF-κB, and RCAN1 can interact with IκBα to inhibit the phosphorylation of IκBα-Y42, increasing the protein level of IκBα We find that overexpression RCAN1 can inhibit the cell viability of lymphoma cell line Raji, increases the endogenous IκBα. level, and increase the Caspase3/7 level to promote cell apoptosis. Meanwhile RCAN1 can inhibit the growth of lymphoma xenograft tumor in SCID mice。 The effect of RCAN1 on inhibiting the activity of NF-κB is independent on NFAT. The former functional area is the N-terminal RCAN1-1-103, and the latter is C-terminal RCAN140-197. Our reaches provided a new method for the treatment of lymphoma, via overexpression RCAN1-1-103 or active endogenous RCAN1 to reduce the sustained activated NF-κB. The application of RCAN1-1-103 can inhibit the cell viability of lymphoma cells and avoid the side-effect caused by RCAN1 inhibiting Calcineurin, and our further will focus on the application of RCAN1-1-103.