Regulation of expression of stromal-derived factor-1 receptors: CXCR4 and CXCR7 in human rhabdomyosarcomas

Abstract

Rhabdomyosarcomas (RMS) express CXCR4 and CXCR7 receptors that bind prometastatic alpha-chemokine stromal-derived factor-1 (SDF-1). In this report, we analyzed the activity of both promoters in a model of less metastatic human embryonal-RMS cell line (RD) and more metastatic alveolar-like RMS (RD cells transduced with paired box gene 3/forkhead homologue; PAX3-FKHR fusion gene). First, CXCR4 is barely detectable in RD and becomes upregulated in RD/PAX3-FKHR cells. In contrast, CXCR7 highly expressed in RD becomes downregulated in RD/PAX3-FKHR cells. Next, promoter deletion and mutation studies revealed that whereas (a) expression of CXCR4 in RD and RD/PAX3-FKHR cells required nuclear respiratory factor-1 (NRF-1) binding site and (b) was additionally upregulated by direct interaction of NRF-1 with PAX3-FKHR, CXCR7 promoter activity required a proximal nuclear factor-kappaB-binding motif. The requirement of these factors for CXCR4 and CXCR7 promoter activities was additionally supported after blocking NRF-1 and nuclear factor-kappaB. Furthermore, CXCR4 expression in PAX3-FKHR(+) RMS cells seems to be enhanced because of the interaction of PAX3-FKHR and NRF-1 proteins in the proximal part of the promoter that prevents access of the negative regulator of transcription YY1 to its binding site. Finally, although hypoxia enhances CXCR4 and CXCR7 promoter activity and receptor expression in RD cells, it inhibits CXCR7 expression in RD/PAX3-FKHR cells. In conclusion, SDF-1 binding receptors CXCR4 and CXCR7 are differently regulated in RMS cells. The upregulation of CXCR4 and downregulation of CXCR7 expression by PAX3-FKHR or hypoxia may give SDF-1 an advantage to better engage the CXCR4 receptor, thus increasing RMS motility.

Figure 1. Panel A. CXCR4 and CXCR7 membrane expression in RMS cell lines RD and RD/PAX3-FKHR

Flow cytometry analysis of membrane expression was performed on the RD cell line transfected with a PAX3-FKHR hybrid construct named RD/PAX3-FKHR and on the RD cell line transfected with empty vector. Percentage of positive cells was calculated on the basis of adequate isotype control (<1%). MFI – Mean Fluorescence Intensity ± SD. The experiment was repeated three times with similar results. A representative study is shown. Panel B. RQ-PCR analysis of CXCR4 and CXCR7 mRNA expression in RD and RD/PAX3-FKHR cell lines. The mRNA expression was measured by Real-Time PCR. Fold of difference was calculated on the basis of 2ΔCt values, where CXCR4 expression in RD cells =1 and CXCR7 expression in RD/PAX3-FKHR =1.

Figure 2. CXCR4 promoter deletion studies and transcription binding sites analysis

Panel A. Constructed CXCR4 promoter inserts. The promoter region of the CXCR4 gene from −2,237 to +62 relative to the start of transcription was cloned and is described as Fragment 1. Fragment 1 was sequentially shortened according to the positions of PAX3 binding sites, and NRF-1 binding sites as depicted in the theoretical model of the cloned sequences of the promoter. The position of PAX3-, NRF-1- HRE-, NF-κb and YY1 binding sites as well as P1-3 primer pairs used in the ChIP experiment are shown. Panel B. CXCR4 promoter activity studies. RD and RD/PAX3-FKHR cells were transfected with the appropriate plasmids. Cultured cells were harvested after 24 hours and assayed for the amount of luciferase activity. Activity was measured on the basis of firefly/Renillla luciferase activity and then the equimolar fold of difference was counted. Results are expressed relative to a value of 1.0 for cells transfected with pGL4.72 vector and empty pGL4.10. Averages of duplicates from three independent experiments are shown. Values are given as the mean ± SEM. Frag1–7 p<0.05, as compared with empty vector. Fragments indicated with asterisk (*), p<0.05, as compared with NRF-1mut fragments and Frag8 (Mann-Whitney test). Panel C. The PAX3-FKHR-NRF-1 complex binds DNA. The ChIP assay was performed on RD/PAX3-FKHR nuclear extracts pulled down with the anti PAX3/7 Ab and revealed two of the PAX3 binding sites assayed were preserved and that the NRF-1 binding site was preserved as well. Moreover, pulling down with NRF Ab resulted in preservation of both PAX3 binding sites. In the RD cell line, beside the NRF binding site, one PAX3 binding site seems to be occupied by transcription factors. No complex formation was visible in RD cells. A representative example is shown from three independent experiments.

Figure 3. PAX3-FKHR binds NRF-1

The NRF-1 binding site was shown to be critical for the increase in CXCR4 promoter activity. Panel A - Western blot analysis of RD and RD/PAX3-FKHR nuclear extracts probed with an anti PAX3/PAX7 Ab (Santa Cruz Biotechnology). We found a band corresponding to wild type PAX3 at 56kD in both cell lines; however, we also found a band at 97kD corresponding to PAX3-FKHR fusion protein. Panel B - Western blot analysis of whole RD and RD/PAX3-FKHR extracts probed with an anti-NRF-1 Ab revealed the presence of a band at 68kD corresponding to the predicted size of the NRF-1 protein. Panel C - an immunoprecipitation with both RD and RD/PAX3-FKHR whole cell extracts with the PAX3/7 Ab. The blot was subsequently probed with the anti NRF-1 Ab. We noticed a band at 68kD in the blot of RD/PAX3-FKHR but not RD immunoprecipitates corresponding to the size of NRF-1. Panel D - reverse experiment, immunoprecipitation with NRF-1 and PAX3 detection shows interaction between NRF-1 and fusion protein PAX3-FKHR. IgG represents the anti-rabbit IgG control for both cell lines.

Figure 4. CXCR7 promoter deletion studies and transcription binding sites analysis

Panel A. CXCR7 promoter inserts. The promoter region of the CXCR7 gene from −2,409 to +89 relative to the start of transcription was cloned and is described as Fragment 1. Fragment 1 shorter derivatives were obtained according to the positions of NRF-1, HRE binding sites, and NF-κB binding sites. The individual constructs were cloned to pGL4.10 vector, transfected into either RD or RD/PAX3-FKHR cells, and used in Dual Luciferase assays. (X) – mutated NF-κB and HRE binding sites. Primer pairs used in the ChIP experiment (N1–N5) flanked 5 different potential binding sites for NF-κB transcription factor. Panel B. NF-κB as a crucial transcription factor that drives CXCR7 promoter activity. Activity of particular CXCR7 promoter constructs was assayed by Dual Luciferase showing an important role of NF-κB transcription factors. Activity was measured on the basis of firefly/Renillla luciferase activity and then the equimolar fold of difference was counted. Results are expressed relative to a value of 1.0 for cells transfected with pGL4.72 vector and empty pGL4.10. Averages of duplicates from three independent experiments are shown. Values are given as the mean ± SEM. Frag 1–6 p<0.05, as compared with empty vector. Fragments indicated with asterisk (*), p<0.05, as compared with NF-κbmut fragment and Frag7 (Mann-Whitney test). Panel C. NF-κB to CXCR7 promoter. ChIP assays of indicated cell lines showed that binding sites 0.03kb, 0.3kb, and 1.0kb are required for full CXCR7 promoter activity. NF-κB-based regulation of the promoter seems to be identical in both cell lines.

Figure 5. YY1 transcription factor negatively regulates CXCR4 and CXCR7 expression

Panel A. PAX3-FKHR translocation upregulates YY1 mRNA expression. RQ-PCR shows ~5-fold upregulation of YY1 mRNA expression after transfection with PAX3-FKHR construct. Panels B and C. YY1 transcription factor is responsible for downregulation of CXCR4 and CXCR7 expression. Both cell lines were transiently transfected with wild type CXCR4 (B) and CXCR7 (C) promoter fragments and fragments containing YY1 mutations. After 24 hours, luciferase activity was measured and promoter activity was calculated as in Figure 2B and Figure 4B. (*) = p<0.05 (paired Student’s t test).

Figure 6. Panel A. Effects of hypoxia on CXCR4 (left) and CXCR7 receptor (right) membrane expression in RD and RD/PAX3-FKHR cell lines

Hypoxia enhances expression of CXCR4 in both RD and RD/PAX3-FKHR cells. At the same time CXCR7 is upregulated during hypoxia in RD/PAX3-FKHR cells, but downregulated in RD cell. Results are the mean of three experiments. Asterisks (*) indicate p<0.05 versus cells cultured in normoxia (paired Student’s t test). MFI – Mean Fluorescence Intensity ±SD. Panel B. YY1 binding to CXCR4 and CXCR7 promoters is divergently regulated by hypoxia. RD and RD/PAX3-FKHR cells were subjected to normoxia and hypoxia for 16 hours and subsequent ChIP assays were performed. PCR products were visualized on gels. Panel C. CXCR4 expression in hypoxia is dependent on HIF-1a. Sequential deletions of CXCR4 promoter reveals the vast importance of the HRE binding site located 1.3kb from the transcriptional start. When a promoter fragment devoid of this HRE was used, promoter activity was abolished. Fold of difference was calculated as in Figure 2B. Values are given as the mean ± SEM, *Activities of Frag3.1, Frag4, and HREmut, p<0.05, as compared with full length promoter fragments (Mann-Whitney test). Panel D. CXCR7 expression in hypoxia is dependent on HIF-1a only in the RD/PAX3-FKHR cell line. Specific constructs lacking HRE binding sites were ligated with pGL4 vector and luciferase activity was measured. Depicted is the fold difference of activity relative to Renilla vector and control. Fold of difference was calculated exactly as in Figure 2B. Values are given as the mean ± SEM. *Activities of Frag6 and HREmut, p<0.05, as compared with full length promoter fragments (Mann-Whitney test).

Figure 7. Effect of hypoxia on chemotactic response of RD (A) and RD/PAX3-FKHR (B) cells

Cells were incubated for 16 hours in hypoxia in the presence of either SDF-1 (300ng/mL) or I-TAC (100ng/mL). Results are mean data of three experiments, * = p<0.05 versus cells cultured in normoxia (paired Student’s t test).