ASSESSMENT OF HUMAN ACKR4 GENE EXPRESSION IN HUMAN EMBRYONIC KIDNEY (HEK) CELLS By: Bahareh Parsi

Chemokines: Chemo-attractant cytokines are highly basic proteins consisting of 70-125 amino acids with molecular masses ranging from 6-14 kD.

Chemokines: Beyond their pivotal role in the coordinated migration of immune cells to the site of inflammation, chemokines are now appreciated to play important roles in the development of lymphoid tissues, in the maturation of immune cells, and in the generation and delivery of adaptive immune responses. Dysregulated expression of chemokines and their corresponding receptors is implicated in a broad range of human diseases, including autoimmune and inflammatory diseases and cancer.

Classical Signaling Chemokine Receptors Chemokine receptors belong to the GPCRs superfamily, and are constituted by single polypeptide chains. Biochem. J; 2008. 409, 635–649 CCR1–10 1. CC chemokines CXCR1–7 2. CXC chemokines 4. C chemokines Single receptors 3. CX3C chemokine CX3CR

Chemokines and cancer Cancer cells were found to express chemokine receptors in a nonrandom manner:

Atypical Chemokine Receptors (ACKRs) Nature Reviews Immunology ;2013.13: 815-829.

ACKR4 (CCRL1) Formerly known as CCRL1, CCR11 and CCX-CKR. ACKR4 binds three homeostatic chemokines, CCL19, CCL21 and CCL25. ACKR4 was shown to bind the homeostatic CXC chemokine, CXCL13, albeit with lower affinity. In brain and testis, an alternative mRNA form of ACKR4 by the human chromosome 3 ACKR4 gene exists, with a unique 5’untranslated region

Expression The expression pattern of ACKR4 in normal human and murine tissues is not entirely clear. In EGFP-reporter mice the ACKR4 expression was shown to be restricted to thymic epithelial cells, lymph vessels of the gut, lymph node stromal cells and keratinocytes in the skin. Other reports indicate a much broader expression (at the mRNA level), including on subsets of leukocytes and in the intestine, heart and lung.

Function: Chemokine scavenging: ACKR4 can internalize and degrade its ligands. Dimerization: ACKR4 forms complexes with signaling chemokine receptors, and inhibits signaling receptor-induced chemotaxis. Chemokine internalization and transcytosis

Cont… ACKR4 and adaptive immunity : ACKR4 ligands (CCL19, CCL21 and CCL25), direct thymocyte navigation and control the maturation and the selection processes that occur in thymus. ACKR4‑deficient mice have been found to spontaneously develop autoimmune disease, CCL25 dysregulation, fewer migratory DCs in the steady state in lymph nodes (Bunting, M. D. et al. 2013). ACKR4 in cancer Feng et al showed for the first time that ACKR4 is a negative regulator of growth and metastasis in breast cancer (Feng et al. 2009 ). In addition,Whether or not heteromerization of ACKR4 with other chemokine receptors is also involved in inhibition of chemotaxis and proliferation of breast cancer remains to be established (Vinet et al. 2014).

The aim of this project was the construction of a vector containing coding sequence of ACKR4 (CCRL1) gene, generation of transient transfectants in HEK293T, and assessment the expression of ACKR4 protein by RT-PCR, flow- cytometry, dot blot and western blot post-transfection.

Materials and methods, Results

Production of recombinant DNA For amplification of human ACKR4 gene, the PCR reaction was done. Restriction digestion: Digestion of PCR product and recipient plasmid with selected enzymes (HindIII and XbaI) Gel purification: agarose gel cut the band under UV light purification with Fermentas kit Ligation: Ligation of DNA fragments by T4 DNA ligase (Bioron),ito produce recombinant molecules

Amplification of ACKR4 and construction of p3XFLAG-Myc-CMV™-26-ACKR4: Results of Gel extraction of p3XFLAG-Myc-CMV™-26 vector and ACKR4 gene after double digestion. 1 2 M 1000 6000 PCR amplification of ACKR4 1000 M 1 p3XFLAG-Myc-CMV™-26 purification by fermentas GeneJET Plasmid Miniprep Kit. 6000 6000 Digestion with HindIII and XbaI

Transformation of bacterial cells : The recombinant DNA molecules were inserted into competent E. coli strain DH5α cells and the bacteria containing the plasmids were selected by their resistance to Ampicillin. Transfection of HEK293T cells: For transfection with Lipofectamine2000 CD reagent, cells were incubated with previously prepared liposomes containing DNA in DMEM medium and after six hours, the medium was changed to DMEM containing 10% FBS. RNA and protein extraction: After homogenizing the cell sample with TRIzol LS reagent, chloroform was added, and the homogenate was allowed to separate into a clear upper aqueous layer (containing RNA) and a lower organic layer (containing the DNA and proteins).

PCR screening of bacterial transformants containing cloned inserts (Colony PCR) 1% agarose gel (100V) for colony PCR of DH5α transformants using Taq DNA polymerase. 5 colonies were screened. Lane 3-7 are transformants from the construction. Lane 1 is the negative control. Expected band size was 1068bp. Gene specific primers were used. 2μL of GeneRuler 1kb Plus DNA Ladder was loaded. 1000 1000bp M 1 2 3 4 5 6

Confirm transformation by isolating the plasmid and performing restriction analysis 1% agarose gel electroforetic analysis of p3XFLAG-Myc-CMV™-26-ACKR4 digested with HindIII /XbaI. Lane 1: digested p3XFLAG-Myc-CMV™-26 by HindIII Lane 2: 1kb DNA molecular weight marker Lane 3: digested construct by HindIII Lane 4: digested construct by HindIII /XbaI showing insert release. 6000bp 8000bp 1 2 3 4 isolation of p3XFLAG-Myc-CMV™-26-ACKR4 plasmid from recombinant E. coli cultures by fermentas GeneJET Plasmid Miniprep Kit. 8000bp 6000bp 7337bp M 1

RT-PCR 1. RNA sample quality 2. cDNA Synthesis 3. PCR Amplification of First Strand cDNA Mix total RNA and random hexamer primer in 12µl volume Add buffer, RNase Inhibitor, dNTPs, Reverse Transcriptase incubate 5min at 25°C, followed by 60min at 42°C and 70°C for 5min

RNA extraction and RT-PCR RNA isolation from transfected HEK293T cells. 28 S 18 S 1000bp M 1 2 3 4 RT-PCR analysis of ACKR4 expression in HEK293T cells. Lane 1: No Template Control Lane 2: No Reverse Transcription control (RT) Lane 4: Mock transfection control RT-PCR product comprising the GAPDH cDNA which is shown by arrowhead. 500bp 500bp

Flow cytometry analysis Following 48h of transfection the cells were released with 0.05 % trypsin-EDTA. the intact cells were treated with DYKDDDDK tag rabbit antibody (Cell Signaling), followed by incubation with FITC-conjugated anti-rabbit IgG antibody (Sigma), and flow cytometry analysis was performed. Dot blot analysis: Dot Blot is a quick technique for detecting, analyzing, and identifying proteins, similar to the western blot technique but differing in that protein samples are not separated electrophoretically. This assay was employed to determine if antibodies and detection system are effective.

Western blot analysis The separated proteins by SDS-PAGE were blotted onto nitrocellulose membrane . At the final stage in western and dot blot, the membrane was treated using BM Chemiluminescence Western Blotting Kit (Roche) and placed in darkness to develop the protein band.

Flow cytometry analysis of ACKR4 expression Unstained histogram IgG Isotype control-FITC histogram Mock transfected cells. Expressing DYKDDDDK-tagged ACKR4 cells. FITC-Conjugated Secondary Antibody Unconjugated, Primary Antibody Sample  1 + Isotype control antibody 2 DYKDDDDK tag antibody 3 4

Western blot and dot blot analysis 1 2 3 Dot blot analysis of extracts from HEK293T cells, mock transfected (lane 1) or expressing DYKDDDDK-tagged ACKR4 (lane2), using DYKDDDDK Tag Antibody. Lane 3: secondary antribody as a positive control 42 kDa 52 kDa Western blot analysis of extracts from HEK293T cells, mock transfected (lane 1) or expressing DYKDDDDK-tagged ACKR4 (lane2), using DYKDDDDK Tag Antibody. 1 2

Conclusion

Though detailed structure–function analysis are not available yet, emerging evidences suggest that chemokine decoy receptors are not “silent,” but they activate G protein independent signaling pathways (Watts et al. ‎2013). the loss of ACKR4 occurs during the development of tumorigenesis and expression of ACKR4 can predict better outcomes (Feng et al. 2009). The inhibition of chemokine receptor-mediated chemotaxis by ACKR4 co-expression may be part of the down regulatory program of immune cells (Vinet et al. 2013). Thus ACKR4 apart from being a scavenger for chemokines may have direct influence on the activity status and chemotactic properties of immune cells. controlled prospective study is desirable to clarify the role of ACKR4 in human cancers and it may provide a novel molecular target in cancer therapy. Therefore, further investigation is required to characterize the functional significance of ACKR4 which might provide an opportunity for therapeutic intervention.

In this study a DNA fragment with the amino acid coding sequence of ACKR4 was cloned into p3XFLAG-Myc-CMV™-26 Expression Vector and generation of transfectants in HEK293T cells, as a first step to understanding the molecular mechanisms of ACKR4 action, was done. HEK293 cells have been extensively used for functional analysis of chemokine receptors. Transfection of HEK293T cells with the FLAG-ACKR4 construct resulted in cell-surface expression of the receptor as examined by flow cytometry, dot lot and western blot analysis following interaction with anti-flag antibody. As the FLAG epitope did not detectably modify the ACKR4 function (Gosling et al. 2000 ) this tag was used for detection of the protein.

by using DYKDDDDK Tag Antibody and goat anti-rabbit IgG-FITC secondary antibody, the expressing transient transfectants were assessed by flow cytometry. As Weak Fluorescence Intensity was observed, there is 3 possible reasons: 1. Target protein expressed at low level. 2. The primary antibody does not work optimally in flow cytometry application. 3. in the sample preparation step, cells were harvested with 500µl of trypsin–EDTA (0.05% ). It is possible that trypsinization also degraded surface antigen.

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