Cluster of differentiation 44 promotes osteosarcoma progression in mice lacking the tumor suppressor Merlin.

Merlin is a versatile tumor suppressor protein encoded by the NF2 gene. Several lines of evidence suggest that Merlin exerts its tumor suppressor activity, at least in part, by forming an inhibitory complex with cluster of differentiation 44 (CD44). Consistently, numerous NF2 mutations in cancer patients are predicted to perturb the interaction of Merlin with CD44. We hypothesized that disruption of the Merlin-CD44 complex through loss of Merlin, unleashes putative tumor- or metastasis-promoting functions of CD44. To evaluate the relevance of the Merlin-CD44 interaction in vivo, we compared tumor growth and progression in Cd44-positive and Cd44-negative Nf2-mutant mice. Heterozygous Nf2-mutant mice were prone to developing highly metastatic osteosarcomas. Importantly, while the absence of the Cd44 gene had no effect on the frequency of primary osteosarcoma development, it strongly diminished osteosarcoma metastasis formation in the Nf2-mutant mice. In vitro assays identified transendothelial migration as the most prominent cellular phenotype dependent on CD44. Adhesion to endothelial cells was blocked by interfering with integrin α4ß1 (very late antigen-4, VLA-4) on osteosarcoma cells and CD44 upregulated levels of integrin VLA-4 ß1 subunit. Among other putative functions of CD44, which may contribute to the metastatic behavior, the passage through the endothelial cells also appears to be critical in vivo, as CD44 significantly promoted formation of lung metastasis upon intravenous injection of osteosarcoma cells into immunocompromised mice. Altogether, our results strongly suggest that CD44 plays a metastasis-promoting role in the absence of Merlin.

Gamma secretase dependent release of the CD44 cytoplasmic tail upregulates IFI16 in cd44-/- tumor cells, MEFs and macrophages.

The adhesion molecule and co-receptor of receptor tyrosine kinases, CD44, is expressed in all cells of the immune system, but also in numerous non-immune cells. CD44 plays roles in the cellular response to different pathogens. The molecular actions of CD44 during these processes are by and large still unknown. The CD44 molecule undergoes a sequential proteolytic cleavage which leads to the release of a soluble intracellular domain (CD44-ICD). Previous reports had shown that the CD44-ICD is taken up into the nucleus where it enhances transcription of specific target genes. By RNA profiling we identified a CD44-dependent transcriptional increase of interferon-responsive genes, among them IFI16. IFI16 is important in the innate immune response. It senses and binds pathogenic DNA and, together with cGAS, activates the cGAS-cGAMP-STING pathway and induces the expression of genes relevant for the response, e.g. IFN-ß. Our results show that the enhancement of IFI16 expression depended on CD44 cleavage. A CD44-negative tumor cell line, embryonic fibroblasts and bone marrow-derived macrophages from cd44-/- mice were reduced in their response to IFN-γ, to viral DNA fragments and to Listeria monocytogenes infection. We could rescue the deficiency of CD44 negative RPM-MC cells and cd44-/- MEFs by expressing only the soluble CD44-ICD in the absence of any other CD44 domain. Expression of the CD44-ICD carrying a mutation that prevented the uptake into the nucleus, could not rescue the absence of CD44. This molecular aspect of regulation by CD44 may explain part of the immune phenotypes of mice with cd44 gene disruption.

Liver Cancer Initiation Requires p53 Inhibition by CD44-Enhanced Growth Factor Signaling.

How fully differentiated cells that experience carcinogenic insults become proliferative cancer progenitors that acquire multiple initiating mutations is not clear. This question is of particular relevance to hepatocellular carcinoma (HCC), which arises from differentiated hepatocytes. Here we show that one solution to this problem is provided by CD44, a hyaluronic acid receptor whose expression is rapidly induced in carcinogen-exposed hepatocytes in a STAT3-dependent manner. Once expressed, CD44 potentiates AKT activation to induce the phosphorylation and nuclear translocation of Mdm2, which terminates the p53 genomic surveillance response. This allows DNA-damaged hepatocytes to escape p53-induced death and senescence and respond to proliferative signals that promote fixation of mutations and their transmission to daughter cells that go on to become HCC progenitors.

Glucocorticoids enhance intestinal glucose uptake via the dimerized glucocorticoid receptor in enterocytes.

Glucocorticoid (GC) treatment of inflammatory disorders, such as inflammatory bowel disease, causes deranged metabolism, in part by enhanced intestinal resorption of glucose. However, the underlying molecular mechanism is poorly understood. Hence, we investigated transcriptional control of genes reported to be involved in glucose uptake in the small intestine after GC treatment and determined effects of GC on electrogenic glucose transport from transepithelial currents. GR(villinCre) mice lacking the GC receptor (GR) in enterocytes served to identify the target cell of GC treatment and the requirement of the GR itself; GR(dim) mice impaired in dimerization and DNA binding of the GR were used to determine the underlying molecular mechanism. Our findings revealed that oral administration of dexamethasone to wild-type mice for 3 d increased mRNA expression of serum- and GC-inducible kinase 1, sodium-coupled glucose transporter 1, and Na(+)/H(+) exchanger 3, as well as electrogenic glucose transport in the small intestine. In contrast, GR(villinCre) mice did not respond to GC treatment, neither with regard to gene activation nor to glucose transport. GR(dim) mice were also refractory to GC, because dexamethasone treatment failed to increase both, gene expression and electrogenic glucose transport. In addition, the rise in blood glucose levels normally observed after GC administration was attenuated in both mutant mouse strains. We conclude that enhanced glucose transport in vivo primarily depends on gene regulation by the dimerized GR in enterocytes, and that this mechanism contributes to GC-induced hyperglycemia.

Commensal microflora and interferon-gamma promote steady-state interleukin-7 production in vivo.

IL-7 is a major regulator of lymphocyte homeostasis; however, little is known about the mechanisms that regulate IL-7 production. To study Il7 gene regulation in vivo, we generated a novel IL-7-reporter mouse, which allows the non-invasive quantification of Il7 gene activity in live mice and, additionally, the simultaneous activation/inactivation of target genes in IL-7-producing cells. With these IL-7-reporter mice, we identify thymus, skin and intestine as major sources of IL-7 in vivo. Importantly, we show that IFN-gamma and the commensal microflora promote steady-state IL-7 production in the intestine. Furthermore, we demonstrate that the blockade of IFN-gamma signaling in intestinal epithelial cells strongly reduces their IFN-gamma-driven IL-7 production. In summary, our data suggest a feedback loop in which commensal bacteria drive IFN-gamma production by lymphocytes, which in turn promotes epithelial cell IL-7 production and the survival of IL-7-dependent lymphocytes.