Mammalian heat shock genes are regulated at the transcriptional level by heat shock factor-1 (HSF-1), a sequence-specific transcription factor. We have examined the role of serine phosphorylation of HSF-1 in the regulation of heat shock gene transcription. Our experiments show that mitogen-activated protein kinases (MAPKs) of the ERK-1 family phosphorylate HSF-1 on serine residues and repress the transcriptional activation of the heat shock protein 70B (HSP70B) promoter by HSF-1 in vivo. These effects of MAPK are transmitted through a specific serine residue (Ser-303) located in a proline-rich sequence within the transcriptional regulatory domain of human HSF-1. However, despite the importance of Ser-303 in transmitting the signal from the MAPK cascade to HSP70 transcription, there was no evidence that Ser-303 could be phosphorylated by MAPK in vitro, although an adjacent residue (Ser-307) was avidly phosphorylated by MAPK. Further studies revealed that Ser-303 is phosphorylated by glycogen synthase kinase 3 (GSK3) through a mechanism dependent on primary phosphorylation of Ser-307 by MAPK. Secondary phosphorylation of Ser-303 by GSK3 may thus repress the activity of HSF-1, and its requirement for priming by MAPK phosphorylation of Ser-307 provides a potential link between the MAPK cascade and HSF-1. Our experiments thus indicate that MAPK is a potent inhibitor of HSF-1 function and may be involved in repressing the heat shock response during normal growth and development and deactivating the heat shock response during recovery from stress.

Rev-Erbalpha (NR1D1) is an orphan nuclear receptor encoded on the opposite strand of the thyroid receptor alpha gene. Rev-Erbalpha mRNA is induced during adipocyte differentiation of 3T3-L1 cells, and its expression is abundant in rat adipose tissue. Peroxisome proliferator-activated receptor gamma (PPARgamma) (NR1C3) is a nuclear receptor controlling adipocyte differentiation and insulin sensitivity. Here we show that Rev-Erbalpha expression is induced by PPARgamma activation with rosiglitazone in rat epididymal and perirenal adipose tissues in vivo as well as in 3T3-L1 adipocytes in vitro. Furthermore, activated PPARgamma induces Rev-Erbalpha promoter activity by binding to the direct repeat (DR)-2 response element Rev-DR2. Mutations of the 5' or 3' half-sites of the response element totally abrogated PPARgamma binding and transcriptional activation, identifying this site as a novel type of functional PPARgamma response element. Finally, ectopic expression of Rev-Erbalpha in 3T3-L1 preadipocytes potentiated adipocyte differentiation induced by the PPARgamma ligand rosiglitazone. These results identify Rev-Erbalpha as a target gene of PPARgamma in adipose tissue and demonstrate a role for this nuclear receptor as a promoter of adipocyte differentiation.

Malignant melanoma cells are known to have altered expression of growth factors compared with normal human melanocytes. These changes probably favor tumor growth and progression and influence the tumor environment. The induction of transforming growth factor β1 (TGF-β1), TGF-β2, and TGF-β3 expression in malignant melanoma has been reported before, whereas the expression of related bone morphogenic protein (BMP) molecules has not been analyzed in melanomas until now. Here, we show that BMP4 and BMP7 are up-regulated in nine melanoma cell lines, whereas BMP2 is overexpressed in only two of the analyzed cell lines. Immunohistochemistry of primary and metastatic melanoma also shows increased BMP4 and BMP7 expression compared with nevi. Promoter studies reveal that expression is controlled at the transcriptional level. The transcription factor Ets-1 was identified as a positive regulator for BMP4 expression. In order to determine the functional relevance of BMP expression in malignant melanoma, chordin-expressing cell clones and antisense BMP4 cell clones were generated. The clones in which BMP4 activity and expression are reduced show no changes in proliferation or in attachment-independent growth when compared with controls. However, a strong reduction of migratory and invasive properties was observed in these cells, suggesting that BMP4 promotes melanoma cell invasion and migration and therefore has an important role in the progression of malignant melanoma.

Heat shock factor 1 (HSF1) is the key transcriptional regulator of the heat shock genes that protect cells from environmental stress. However, because heat shock gene expression is deleterious to growth and development, we have examined mechanisms for HSF1 repression at growth temperatures, focusing on the role of phosphorylation. Mitogen-activated protein kinases (MAPKs) of the ERK family phosphorylate HSF1 and represses transcriptional function. The mechanism of repression involves initial phosphorylation by MAP kinase on serine 307, which primes HSF1 for secondary phosphorylation by glycogen synthase kinase 3 on a key residue in repression (serine 303).In vivo expression of glycogen synthase kinase 3 (α or β) thus represses HSF1 through phosphorylation of serine 303. HSF1 is also phosphorylated by MAPK in vitro on a second residue (serine 363) adjacent to activation domain 1, and this residue is additionally phosphorylated by protein kinase C. In vivo, HSF1 is repressed through phosphorylation of this residue by protein kinase Cα or -ζ but not MAPK. Regulation at 37 °C, therefore, involves the action of three protein kinase cascades that repress HSF1 through phosphorylation of serine residues 303, 307, and 363 and may promote growth by suppressing the heat shock response.

Angiogenesis is the formation of new capillaries from pre-existing blood vessels and participates in proper vasculature development. In pathological conditions such as cancer, abnormal angiogenesis takes place. Angiogenesis is primarily carried out by endothelial cells, the innermost layer of blood vessels. The vascular endothelial growth factor-A (VEGF-A) and its receptor-2 (VEGFR-2) trigger most of the mechanisms activating and regulating angiogenesis, and have been the targets for the development of drugs. However, most experimental assays assessing angiogenesis rely on animal models. We report an in vitro model using a microvessel-on-a-chip. It mimics an effective endothelial sprouting angiogenesis event triggered from an initial microvessel using a single angiogenic factor, VEGF-A. The angiogenic sprouting in this model is depends on the Notch signaling, as observed in vivo. This model enables the study of anti-angiogenic drugs which target a specific factor/receptor pathway, as demonstrated by the use of the clinically approved sorafenib and sunitinib for targeting the VEGF-A/VEGFR-2 pathway. Furthermore, this model allows testing simultaneously angiogenesis and permeability. It demonstrates that sorafenib impairs the endothelial barrier function, while sunitinib does not. Such in vitro human model provides a significant complimentary approach to animal models for the development of effective therapies.

The recruitment and proliferation of smooth muscle cells and pericytes are two key events for the stabilization of newly formed capillaries during angiogenesis and, when out of control in the adult, are the main causes of arteriosclerosis. We have identified a novel gene, named VE‐statin for vascular endothelial‐statin, which is expressed specifically by endothelial cells of the developing mouse embryo and in the adult, and in early endothelial progenitors. The mouse and human VE‐statin genes have been located on chromosome 2 and 9, respectively, they span >10 kbp and are transcribed in two major variants arising from independent initiation sites. The VE‐statin transcripts code for a unique protein of 30 kDa that contains a signal peptide and two epidermal growth factor (EGF)‐like modules. VE‐statin is found in the cellular endoplasmic reticulum and secreted in the cell supernatant. Secreted VE‐statin inhibits platelet‐derived growth factor (PDGF)‐BB‐induced smooth muscle cell migration, but has no effects on endothelial cell migration. VE‐statin is the first identified inhibitor of mural cell migration specifically produced by endothelial cells.