The NF2 tumor suppressor gene product, merlin, mediates contact inhibition of growth through interactions with CD44.

The neurofibromatosis-2 (NF2) gene encodes merlin, an ezrin-radixin-moesin-(ERM)-related protein that functions as a tumor suppressor. We found that merlin mediates contact inhibition of growth through signals from the extracellular matrix. At high cell density, merlin becomes hypo-phosphorylated and inhibits cell growth in response to hyaluronate (HA), a mucopolysaccharide that surrounds cells. Merlin's growth-inhibitory activity depends on specific interaction with the cytoplasmic tail of CD44, a transmembrane HA receptor. At low cell density, merlin is phosphorylated, growth permissive, and exists in a complex with ezrin, moesin, and CD44. These data indicate that merlin and CD44 form a molecular switch that specifies cell growth arrest or proliferation.

The BRG-1 subunit of the SWI/SNF complex regulates CD44 expression.

Aberrant regulation of CD44, a transmembrane glycoprotein, has been implicated in the growth and metastasis of numerous tumors. Although both CD44 overexpression and loss have been implicated in tumor progression, the mechanism of CD44 down-regulation in these tumor types is not known. By immunoblot and reverse transcription-polymerase chain reaction analysis we determined that a cervical carcinoma cell line, C33A, lacks CD44 expression. To determine how CD44 is down-regulated in C33A cells, we utilized cell fusions of C33A cells with a CD44-expressing cell line (SAOS-2). We found that SAOS-2 fusion restored CD44 expression in C33A cells, suggesting that a trans-acting factor present in SAOS-2 cells promotes CD44 production. C33A cells are BRG-1-deficient, and we found that CD44 was absent in another BRG-1-deficient tumor cell line, indicating that loss of BRG-1 may be a general mechanism by which cells lose CD44. Reintroduction of BRG-1 into these cells restored CD44 expression. Furthermore, disruption of BRG-1 function through the use of dominant-negative BRG-1 demonstrated the requirement of BRG-1 in CD44 regulation. Finally, we show that Cyclin E overexpression resulted in the attenuation of CD44 stimulation, which is consistent with previous observations that Cyclin E can abrogate BRG-1 action. Taken together, these results suggest that BRG-1 is a critical regulator of CD44 expression, thus implicating SWI/SNF components in the regulation of cellular adhesion and metastasis.

Positive and negative elements modulate the promoter of the human liver-specific alpha2-HS-glycoprotein gene.

The human alpha2-HS-glycoprotein (AHSG) and the 63-kDa rat phosphoprotein (pp63) are homologous plasma proteins that belong to the fetuin family. AHSG and pp63 are involved in important functions such as inhibition of insulin receptor tyrosine kinase activity, inhibition of protease activities, and regulation of calcium metabolism and osteogenesis. Studies of the AHSG proximal promoter performed in vitro in rat and human cells indicate that several NF-1 and C/EBP binding sites exert a positive effect on its transcriptional activity. However, until now, no distal elements have been examined in this gene, in either species. We report that the human AHSG gene promoter acts in a liver-specific manner and is further controlled by three distal, 5'-flanking elements. The negative elements III and I are, respectively, located 5' and 3' of the positive element II. All three elements require the natural context of the human AHSG gene to fully exert their negative or positive effect. Element I harbours a single binding site for NF-1. This nuclear factor thus appears to be able to up- or downregulate the AHSG gene depending on the site it binds to. Elements I, II and possibly III are absent in the rodent Ahsg gene encoding pp63.

Down-regulation of negative acute-phase response genes by hypotonic stress in HepG2 hepatoma cells.

An increased hepatocellular hydration state (HS) that can be induced by hypotonic stress or a high glutamine uptake modulates the transcription of given genes in liver. This could be important in the acute phase (AP) of a systemic inflammation where both HS and glutamine uptake transiently increase in liver. In HepG2 hepatoma cells cultured in conditions of hypotonic stress or a high extracellular glutamine availability, a specifically decreased expression of two human mRNAs, namely those of alphal-microglobulin/bikunin precursor (AMBP) and alpha2-HS-glycoprotein, that are also down-regulated in liver by AP, could be seen. A functional analysis of the AMBP promoter indicated that this hypotonic stress-induced down-regulation takes place at a transcriptional level. In these experiments, the mRNA level and transcription of the glyceraldehyde-3-phosphate dehydrogenase gene that are known to be unmodified in AP did not exhibit any change. Given that hypotonic stress also upregulates the transcription of a liver gene that is also upregulated in AP [Meisse et al. (1998) FEBS Lett. 422, 3463481, the AP-associated increase in hepatocellular HS now appears to participate in the transcriptional control of both sets of genes that are up- or down-regulated in AP.

Structural and functional analysis of the 5'-transcription control region for the human alpha2-HS glycoprotein gene.

The human alpha2-HS glycoprotein (A2HS) and rat phosphoprotein of Mr 63000 (pp63) are homologous plasma proteins and members of the fetuin superfamily. A2HS is involved in important functions such as inhibition of the tyrosine kinase activity of the insulin receptor, regulation of calcium metabolism and osteogenesis as well as protease inhibitory activity. We report an analysis of the 5' transcription control region (4 kb) of the A2HS gene. Its most proximal 300 nt display a very potent transcriptional activity. The latter is likely accounted for by C/EBP and NF1 binding sites that are conserved from the human A2HS gene to the rat pp63 gene. In contrast, these human and rat genes appear to largely diverge beyond their proximal promoter.

Differential expression of cytokine genes in monocytes, peritoneal macrophages and liver following endotoxin- or turpentine-induced inflammation in rat.

Pro-inflammatory cytokines are produced after systemic or local inflammation by a wide variety of cell types including monocytes, macrophages, Kupffer and endothelial cells. Previous studies have shown that IL-6 gene expression does not occur in liver from rats undergoing an acute phase response after turpentine injection or controls. These data do not rule out the possibility that delivery of a pathogen to the liver via the portal circulation could directly activate the Kupffer cells. Rats were injected either intravenously or intraperitoneally with LPS, or subcutaneously with turpentine oil. The changes in IL-1 beta, IL-6, and TNF mRNA levels in monocytes (collected from portal vein or caval cein), peritoneal macrophages and liver over a 3-hour period post-treatment were examined. The kinetics of LPS-vs turpentine-induced cytokine mRNAs in these various cell types were compared by quantitative reverse transcription and polymerase chain reaction (RT-PCR). Our data demonstrate that an intrahepatic expression of cytokines in the non parenchymal cells was induced by an LPS challenge but not by a turpentine-induced inflammation. This process could act as a paracrine mechanism in the acute-phase response and play a role in the modulation of hepatic regeneration.