Megakaryoblastic leukemia 1 (MKL1) is a myocardin-related transcription factor that we found strongly activated serum response element (SRE)-dependent reporter genes through its direct binding to serum response factor (SRF). The c-fos SRE is regulated by mitogen-activated protein kinase phosphorylation of ternary complex factor (TCF) but is also regulated by a RhoA-dependent pathway. The mechanism of this pathway is unclear. Since MKL1 (also known as MAL, BSAC, and MRTF-A) is broadly expressed, we assessed its role in serum induction of c-fos and other SRE-regulated genes with a dominant negative MKL1 mutant (DN-MKL1) and RNA interference (RNAi). We found that DN-MKL1 and RNAi specifically blocked SREdependent reporter gene activation by serum and RhoA. Complete inhibition by RNAi required the additional inhibition of the related factor MKL2 (MRTF-B), showing the redundancy of these factors. DN-MKL1 reduced the late stage of serum induction of endogenous c-fos expression, suggesting that the TCF-and RhoAdependent pathways contribute to temporally distinct phases of c-fos expression. Furthermore, serum induction of two TCF-independent SRE target genes, SRF and vinculin, was nearly completely blocked by DN-MKL1. Finally, the RBM15-MKL1 fusion protein formed by the t(1;22) translocation of acute megakaryoblastic leukemia had a markedly increased ability to activate SRE reporter genes, suggesting that its activation of SRF target genes may contribute to leukemogenesis.

Emerging evidence suggests that the primary tumor influences the development of supportive metastatic microenvironments, referred to as pre-metastatic niches, in certain distant organs before arrival of metastatic cells. However, the mechanisms underlying the contributions of the primary tumor to pre-metastatic niche formation are not fully understood. Here we demonstrate that colorectal carcinoma cells secrete VEGF-A, which stimulates tumor-associated macrophages to produce CXCL1 in the primary tumor. Elevation of CXCL1 in pre-metastatic liver tissue recruited CXCR2-positive myeloid-derived suppressor cells (MDSC) to form a pre-metastatic niche that ultimately promoted liver metastases. Importantly, pre-metastatic liver-infiltrating MDSC induced tumor cell survival without involvement of innate or adaptive immune responses. Our study provides the first evidence that primary malignant cell-secreted VEGF-A stimulates tumor-associated macrophages to produce CXCL1, which recruits CXCR2-positive MDSC to form a pre-metastatic niche to promote liver metastases. Our findings not only shed light on how the tumor microenvironment contributes to pre-metastatic niche formation at distant sites, but they also provide comprehensive insights into how MDSC are recruited to other organs where they contribute to metastatic spread of disease. Moreover, our work also provides a rational for development of CXCR2 antagonists to inhibit or prevent metastatic spread of disease.

The chemokine receptor CXCR4 has recently been shown to be a co-receptor involved in the entry of human immunodeficiency virus type 1 into target cells. This study shows that coexpression of ␤-arrestin with CXCR4 in human embryonic kidney 293 cells attenuated chemokine-stimulated G protein activation and inhibition of cAMP production. Truncation of the C-terminal 34 amino acids of CXCR4 (CXCR4-T) abolished the effects of ␤-arrestin on CXCR4/G protein signaling, indicating the functional interaction of the receptor C terminus with ␤-arrestin. On the other hand, receptor internalization and the subsequent activation of extracellular signal-regulated kinases were significantly promoted by coexpression of ␤-arrestin with CXCR4, whereas the C-terminal truncation of CXCR4 did not affect this regulation of ␤-arrestin, suggesting that ␤-arrestin can functionally interact with CXCR4 with or without the C terminus. Moreover, ␤ 2 V54D, the dominant inhibitory mutant of ␤-arrestin 2, exerted no effects on CXCR4/G protein signaling, but strongly influenced receptor internalization and extracellular signalregulated kinase activation. Further cross-linking experiments demonstrated that ␤-arrestin as well as ␤ 2 V54D could physically contact both CXCR4 and CXCR4-T. Glutathione S-transferase pull-down assay showed that ␤-arrestin was able to bind efficiently in vitro to both the third intracellular loop and the 34-amino acid C terminus of CXCR4. Taken together, our data clearly establish that ␤-arrestin can effectively regulate different functions of CXCR4 and that this is mediated through its distinct interactions with the C terminus and other regions including the third loop of CXCR4.

Myocardin, megakaryoblastic leukemia-1 (MKL1), and MKL2 belong to a newly defined family of transcriptional coactivators. All three family members bind to serum response factor (SRF) and strongly activate transcription from promoters with SRF binding sites. SRF is required for the serum induction of immediate early genes such as c-fos and for the expression of many muscle specific genes. Consistent with a role in muscle specific gene expression, myocardin is specifically expressed in cardiac and smooth muscle cells while MKL1 and 2 are broadly expressed. Myocardin has particularly been shown to be required for smooth muscle development while MKL1/2 are required for the RhoA signaling pathway for induction of immediate early genes. SRF can be activated by at least two families of coactivators, p62TCF and myocardin/MKL. These factors bind to the same region of SRF such that their binding is mutually exclusive. This provides one mechanism of regulation of SRF target genes by pathways that differentially activate the coactivators. The RhoA pathway appears to activate MKL1 by altering MKL1's binding to actin and causing MKL1's translocation from the cytoplasm to the nucleus. However, this mechanism of activation of the myocardin/MKL family has not been observed in all cell types such that other regulatory mechanism(s) likely exist. In particular, rapid serum inducible phosphorylation of MKL1 was observed. The regulation of this coactivator family is key to understanding how SRF target genes are activated during muscle cell differentiation or growth factor induced cell proliferation.