Brg-1 mediates the constitutive and fenretinide-induced expression of SPARC in mammary carcinoma cells via its interaction with transcription factor Sp1

BACKGROUND: Secreted protein, acidic and rich in cysteine (SPARC) is a matricellular protein that mediates cell-matrix interactions. It has been shown, depending on the type of cancer, to possess either pro- or anti-tumorigenic properties. The transcriptional regulation of the SPARC gene expression has not been fully elucidated and the effects of anti-cancer drugs on this process have not been explored. RESULTS: In the present study, we demonstrated that chromatin remodeling factor Brg-1 is recruited to the proximal SPARC promoter region (-130/-56) through an interaction with transcription factor Sp1. We identified Brg-1 as a critical regulator for the constitutive expression levels of SPARC mRNA and protein in mammary carcinoma cell lines and for SPARC secretion into culture media. Furthermore, we found that Brg-1 cooperates with Sp1 to enhance SPARC promoter activity. Interestingly, fenretinide [N-4(hydroxyphenyl) retinamide, 4-HPR], a synthetic retinoid with anti-cancer properties, was found to up-regulate the transcription, expression and secretion of SPARC via induction of the Brg-1 in a dose-dependent manner. Finally, our results demonstrated that fenretinide-induced expression of SPARC contributes significantly to a decreased invasion of mammary carcinoma cells. CONCLUSIONS: Overall, our results reveal a novel cooperative role of Brg-1 and Sp1 in mediating the constitutive and fenretinide-induced expression of SPARC, and provide new insights for the understanding of the anti-cancer effects of fenretinide

Macrophage regulatory genes Nramp1 and MK2 : implication in inflammation and cutaneous wound healing

Macrophages are active participants in many important biological processes, including antimicrobial activity, tumour surveillance, apoptotic cell clearance, homeostasis and wound healing. The activity of all cells is under the direct influence of their genetic makeup and macrophages are no exception. Natural resistance-associated macrophage protein 1 (Nramp1, also known as SLC11A1) is a macrophage-restricted gene that confers resistance to intracellular pathogens in mice. Mitogen activated protein kinase activated protein kinase 2 (MAPKAPK-2 or MK2), a substrate of p38 MAPK, is known to influence the activation of macrophages in response to stressors, including the Toll-like receptor (TLR)-4 ligand LPS. Like NRAMP1, MK2 has also been shown to influence the efficiency of the antibacterial response. The present study evaluates the role of NRAMP1 and MK2 in TLR-mediated cytokine induction and their role in cutaneous wound healing. Mice lacking NRAMP1 are severely impaired in their rate of cutaneous wound healing. Nramp1 gene ablation has been associated with lower levels of SLPI, a protein previously demonstrated to influence the rate of wound healing in a non-redundant fashion. Macrophages derived from Nramp1-null mice are less efficient in activating p38 MAPK signaling, which results in lower levels of MK2 phosphorylation. The reduced level of p38 MAPK and MK2 activation in Nramp1-null macrophages also correlates with decreased cytokine induction in response to TLR7 ligand stimulation of these cells. Using p38 MAPK inhibitor and MK2-deficient macrophages, we demonstrate that TLR7- and TLR9-mediated cytokine induction is directly under the control of this signaling pathway. Furthermore, cytokine induction is regulated by MK2 at the post-transcriptional level. Macrophage-induced cytokines play an important role in cutaneous wound healing. Since MK2-deficient macrophages are severely impaired in their ability to induce cytokines following activation, we next evaluated the role of MK2 in cutaneous wound healing. Our results demonstrate that the rate of wound healing is significantly delayed in the absence of MK2. The level of cytokine expression in the wounds is impaired and macrophages are major players in cutaneous wound healing. Our data also show that intradermal transfer of macrophages with intact MK2 significantly improved wound healing kinetics. Overall, the studies presented in this dissertation demonstrate the importance of NRAMP1 and MK2 in the modulation of macrophage gene expression, and their important role in the control of cutaneous wound healing

c-Src kinase is involved in the tyrosine phosphorylation and activity of SLC11A1 in differentiating macrophages

<div><p>Studies have demonstrated that the solute carrier family 11 member 1 (SLC11A1) is heavily glycosylated and phosphorylated in macrophages. However, the mechanisms of SLC11A1 phosphorylation, and the effects of phosphorylation on SLC11A1 activity remain largely unknown. Here, the tyrosine phosphorylation of SLC11A1 is observed in SLC11A1-expressing U937 cells when differentiated into macrophages by phorbol myristate acetate (PMA). The phosphorylation of SLC11A1 is almost completely blocked by treatment with PP2, a selective inhibitor of Src family kinases. Furthermore, we found that SLC11A1 is a direct substrate for active c-Src kinase and siRNA-mediated knockdown of cellular Src (c-Src) expression results in a significant decrease in tyrosine phosphorylation. We found that PMA induces the interaction of SLC11A1 with c-Src kinase. We demonstrated that SLC11A1 is phosphorylated by Src family kinases at tyrosine 15 and this type of phosphorylation is required for SLC11A1-mediated modulation of NF-κB activation and nitric oxide (NO) production induced by LPS. Our results demonstrate important roles for c-Src tyrosine kinase in phosphorylation and activation of SLC11A1 in macrophages.</p></div

The proline-rich motif of SLC11A1 is required for association with c-Src kinase.

<p>(A) Schematic representation of the P130A, P231A and PRM-deletion constructs of SLC11A1. (B) U937 cells were transfected with constructed pCB6 vectors expressing c-Myc-tagged wild-type SLC11A1, P231A, P130A or PRM-deletion mutant together with c-Src expression vector. Transfected cells were treated with PMA for 48 hrs and cell lysates were immunoprecipitated with anti-c-Myc antibody (9E10). The bound proteins were probed with a rabbit polyclonal antibody against c-Src. Expression of c-Myc-tagged wild-type SLC11A1, P231A, P130A or PRM-deletion mutant in transfected cells were also detected by Western blot analysis. (C) <i>In vitro</i> binding of c-Src to the PXXP motifs of SLC11A1. The SLC11A1 PRM peptide I (GEVCHLYYPKVPRTVLWLTI), PRM peptide II (ISSPTSPTSPGPRQAPPRE T), the negative control peptide (IPDTKPGTFSLRK LWAFTGPGFLM) and the positive control peptide (Src substrate Sam68), which were described in Materials and Methods, were coupled to activated CH-Sepharose 4B. Binding of these peptides to purified c-Src was detected by immunoblotting with anti-Src antibody. A representative Western blot is shown. (D) Peptide competition binding assay was performed. Purified c-Src was added 1, 2 or 5 μg of negative peptide, peptide I or peptide II before incubating with the CH-Sepharose 4B coupled with the positive control peptide. The illustrated showing the inhibitory effects of different peptides on c-Src binding to the positive peptide.</p

Inhibition of Src family kinase activity blocks tyrosine phosphorylation of SLC11A1.

<p>U937-SLC11A1 cells were cultured with PMA (10ng/ml) for 48 hrs and were then left untreated (CTR) or treated with PP2 or PP3 (inactive analogue) for another 24 hours. Cell lysates were prepared. (A) c-Src and active c-Src (pY418) were monitored by Western blotting analysis. (B) Cell lysates were immunoprecipitated with the anti-c-Myc antibody 9E10, and the immunoprecipitates were probed with antibody 4G10 to phosphotyrosine for phosphorylated SLC11A1. The Western blots were stripped and re-probed with an antibody against SLC11A1. (C) The level of SLC11A1 phosphorylation was quantified by densitometry analysis and normalized to the level of total SLC11A1 protein. The relative phosphorylation level of SLC11A1 in untreated control group was set as 100%. The inhibitory effect of PP2 on SLC11A1 phosphorylation was assessed in 3 separate experiments (mean± SE). ***<i>P</i><0.001, compared with untreated control group.</p

<i>In vitro</i> phosphorylation of SLC11A1 by c-Src.

<p>A fixed amount of GST-SLC11A1 (500ng, a.a.1-a.a.178) was incubated with increasing amounts (50-200ng) of purified Src, as indicated. Phosphorylation levels of protein were analyzed by immunoblotting with anti-phosphotyrosine antibodies (4G10) (top). The same blots were then reprobed with anti-GST antibodies for GST-SLC11A1 fusion protein as a loading control.</p

Effect of PMA treatment on the tyrosine phosphorylation of SLC11A1 and Src kinase activity.

<p>U937-SLC11A1 cells (stably expressing SLC11A1-c-Myc fusion protein) were left untreated or treated with PMA (10ng/ml) for 6, 24 and 72 hrs. (A) Cell lysates were immunoprecipitated with an anti-c-Myc antibody (9E10). The phosphorylation levels of SLC11A1 were analyzed by immuoblotting with an antibody to phosphotyrosine (4G10). The same blots were reprobed with an anti-SLC11A1 antibody. (B) Cell lysates were separated on SDS-PAGE and the expression of c-Src protein was detected by Western blot analysis. The blots were stripped and re-probed with an antibody against β-tubulin as a loading control. (C) Cell lysates were immunoprecipated with a specific antibody against c-Src. <i>In vitro</i> kinase activity assay was performed using Sam68 as a substrate (top panel). The immunoprecipitates were also probed with a specific anti-c-Src antibody (bottom panel).</p

The proline-rich motif of SLC11A1 is required for association with c-Src kinase.

<p>(A) Schematic representation of the P130A, P231A and PRM-deletion constructs of SLC11A1. (B) U937 cells were transfected with constructed pCB6 vectors expressing c-Myc-tagged wild-type SLC11A1, P231A, P130A or PRM-deletion mutant together with c-Src expression vector. Transfected cells were treated with PMA for 48 hrs and cell lysates were immunoprecipitated with anti-c-Myc antibody (9E10). The bound proteins were probed with a rabbit polyclonal antibody against c-Src. Expression of c-Myc-tagged wild-type SLC11A1, P231A, P130A or PRM-deletion mutant in transfected cells were also detected by Western blot analysis. (C) <i>In vitro</i> binding of c-Src to the PXXP motifs of SLC11A1. The SLC11A1 PRM peptide I (GEVCHLYYPKVPRTVLWLTI), PRM peptide II (ISSPTSPTSPGPRQAPPRE T), the negative control peptide (IPDTKPGTFSLRK LWAFTGPGFLM) and the positive control peptide (Src substrate Sam68), which were described in Materials and Methods, were coupled to activated CH-Sepharose 4B. Binding of these peptides to purified c-Src was detected by immunoblotting with anti-Src antibody. A representative Western blot is shown. (D) Peptide competition binding assay was performed. Purified c-Src was added 1, 2 or 5 μg of negative peptide, peptide I or peptide II before incubating with the CH-Sepharose 4B coupled with the positive control peptide. The illustrated showing the inhibitory effects of different peptides on c-Src binding to the positive peptide.</p