As shown inFig.1c, the cells stably transfected with CD44pcDNA3.1 expression vector, but not vector control, had the ability to adhere to hyaluronancoated wells. regulating Akt activation. Strikingly, constitutively active Akt rescued the proliferation defect showing requirement for active Akt, in our system. Conclusion:Our results suggest a novel pathway by which Rigosertib CD44 inactivates Akt, downregulates EGR1 expression and inhibits cell proliferation. == Introduction == CD44, a type I transmembrane glycoprotein, has diverse roles in a number of cell functions including: (i) leucocyte trafficking (1), (ii) angiogenesis (2) and (iii) cell proliferation (3,4,5,6). Although CD44 does not contain any signalling domain, it can act as a platform for recruitment and assembly of molecular machinery for signal transduction (7). For example, CD44 clustering in T cells can recruit tyrosine phosphatase CD45 to the CD44 cluster. CD45 then dephosphorylates the negative regulatory tyrosine of Src family kinase, Lck. In turn, this signalling event results in Factin ring formation and round cell spreading (8). A number of reports have shown that CD44 can induce or augment cell proliferative responses. For example, CD44 can trigger mobilization of Ca2+in aortic endothelial cells which in turn leads to their proliferation (9). Other mechanisms whereby Rigosertib CD44 induces cell proliferation have also been reported, including activation of MAP kinases (10). By contrast, negative regulation of cell proliferation by CD44 has been less frequently described with only one report showing that CD44 can inhibit proliferation of the NB4 cell line (3). Thus, it is tempting to speculate that CD44 has the potential to regulate cell proliferation in both positive and negative fashions. The purpose of this study was to evaluate the molecular basis for CD44mediated antiproliferative effects, using the E6.1 Jurkat cell system (11). Importantly, E6.1 Jurkat cells do not express endogenous CD44. Therefore, the molecular and biochemical mechanisms whereby CD44 regulates cellular processes can be directly investigated in E6.1 Jurkat cells expressing CD44, by comparison with their open vector control counterparts (11). Here, we show that CD44 expression in E6.1 Rigosertib Jurkat cells inhibited their proliferation compared to cells transfected with the open vector control. Moreover, CD44 reduced expression of early growth response1 (EGR1) gene. EGR1 protein is a zinc finger transcription factor that can bind and activate promoters of many genes, whose products can influence cell proliferation (12,13,14,15,16). Indeed, transfection of E6.1 Jurkat cells with EGR1 siRNA, but not siRNA control, inhibited cell proliferation, confirming its role in proliferation of these cells. In terms of a mechanism for how CD44 Rigosertib could regulate EGR1, we found that EGR1 expression was regulatedviathe PI3K/Akt pathway in E6.1 Jurkat cells. Moreover, we found that CD44 disrupted Akt activation as assessed by Western blotting and that constitutively active Akt rescued the proliferation defect. Thus, our results suggest a novel pathway in which CD44 can negatively regulate cell proliferationviaAkt inactivation and downregulated EGR1 expression. == Materials and methods == == Cell lines == E6.1 Jurkat cells were purchased from the American Type Culture Collection and were maintained as suggested by the NKSF supplier. The human lymphoma cell line HuT78 was kindly provided by Dr Elisa Fleming (U.T. Southwestern Medical Center, Dallas, TX) and cultured in RPMI supplemented with 10% heatinactivated FBS, 1% sodium pyruvate, 25 mmHEPES and 1% penicillin/streptomycin/glutamine. == Antibodies and reagents == Pan antiCD44 antibody, clone IM7 was from BD Biosciences, San Jose, CA, USA. Goat antihuman EGR1 and rat IgG isotype control were from R&D Systems, Tustin, CA, USA. FITCconjugated antirat secondary antibody was from Caltag Laboratories Inc. (Burlingame, CA, USA) and secondary antibodies labelled with alkaline phosphatise, for Western blotting, were purchased from Invitrogen, Carlsbad, CA, USA. Antibodies against actin, Akt and phosphorylated Akt were all from Cell Signaling (Danvers, MA, USA). Pharmacological inhibitors wortmannin, LY294002, SB239063 and U0126 and bovine testis hyaluronidase (EC 3.2.1.35, type 1S) were from SigmaAldrich (St. Louis, MO, USA). Sodium hyaluronan was from Acros Organics (Geel, Belgium). All SYBRlabelled primers used for RT2PCR were from SABiosciences (Frederick, MD, USA). == Cloning of Rigosertib CD44 == Total RNA was extracted from HuT78 cells using TRIzol Reagent (Invitrogen). Standard form CD44 transcripts were amplified using SuperScript III onestep RTPCR system (Invitrogen), and ahead primer 5CCCAAGCTTGGATCCTCCAGCTCCTTTCG3 comprising an engineeredHindIII site (underlined) and reverse primer 5GCTCTAGATCCCAGCTCCCTGTAATGGT3 comprising engineeredXbaI site (underlined). Biking conditions were as follows: 50 C for 20 min, 94 C for 2.