Increased endothelial cell (EC) permeability is central to the pathophysiology of inflammatory syndromes such as sepsis and acute lung injury (ALI). Activated protein C (APC), a serine protease critically involved in the regulation of coagulation and inflammatory processes, improves sepsis survival through an unknown mechanism. We hypothesized a direct effect of APC to both prevent increased EC permeability and to restore vascular integrity after edemagenic agonists. We measured changes in transendothelial electrical resistance (TER) and observed that APC produced concentration-dependent attenuation of TER reductions evoked by thrombin. We next explored known EC barrier-protective signaling pathways and observed dosedependent APC-mediated increases in cortical myosin light chain (MLC) phosphorylation in concert with cortically distributed actin polymerization, findings highly suggestive of Rac GTPase involvement. We next determined that APC directly increases Rac1 activity, with inhibition of Rac1 activity significantly attenuating APC-mediated barrier protection to thrombin challenge. Finally, as these signaling events were similar to those evoked by the potent EC barrier-enhancing agonist, sphingosine 1-phosphate (S1P), we explored potential cross-talk between endothelial protein C receptor (EPCR) and S1P 1 , the receptors for APC and S1P, respectively. EPCR-blocking antibody (RCR-252) significantly attenuated both APC-mediated barrier protection and increased MLC phosphorylation. We next observed rapid, EPCR and PI 3-kinase-dependent, APCmediated phosphorylation of S1P 1 on threonine residues consistent with S1P 1 receptor activation. Co-immunoprecipitation studies demonstrate an interaction between EPCR and S1P 1 upon APC treatment. Targeted silencing of S1P 1 expression using siRNA significantly reduced APCmediated barrier protection against thrombin. These data suggest that novel EPCR ligation and S1P 1 transactivation results in EC cytoskeletal rearrangement and barrier protection, components potentially critical to the improved survival of APC-treated patients with severe sepsis.

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Endothelial cell (EC) barrier dysfunction results in increased vascular permeability observed in inflammation, tumor angiogenesis, and atherosclerosis. The platelet-derived phospholipid sphingosine-1-phosphate (S1P) decreases EC permeability in vitro and in vivo and thus has obvious therapeutic potential. We examined S1P-mediated human pulmonary artery EC signaling and barrier regulation in caveolin-enriched microdomains (CEM). Immunoblotting from S1P-treated EC revealed S1P-mediated rapid recruitment (1 microM, 5 min) to CEMs of the S1P receptors S1P1 and S1P3, p110 PI3 kinase alpha and beta catalytic subunits, the Rac1 GEF, Tiam1, and alpha-actinin isoforms 1 and 4. Immunoprecipitated p110 PI3 kinase catalytic subunits from S1P-treated EC exhibited PIP3 production in CEMs. Immunoprecipitation of S1P receptors from CEM fractions revealed complexes containing Tiam1 and S1P1. PI3 kinase inhibition (LY294002) attenuated S1P-induced Tiam1 association with S1P1, Tiam1/Rac1 activation, alpha-actinin-1/4 recruitment, and EC barrier enhancement. Silencing of either S1P1 or Tiam1 expression resulted in the loss of S1P-mediated Rac1 activation and alpha-actinin-1/4 recruitment to CEM. Finally, silencing S1P1, Tiam1, or both alpha-actinin isoforms 1/4 inhibits S1P-induced cortical F-actin rearrangement and S1P-mediated barrier enhancement. Taken together, these results suggest that S1P-induced recruitment of S1P1 to CEM fractions promotes PI3 kinase-mediated Tiam1/Rac1 activation required for alpha-actinin-1/4-regulated cortical actin rearrangement and EC barrier enhancement.

The role for hyaluronan (HA) and CD44 in vascular barrier regulation is unknown. We examined high and low molecular weight HA (HMW-HA, ϳ1,000 kDa; LMW-HA, ϳ2.5 kDa) effects on human transendothelial monolayer electrical resistance (TER). HMW-HA increased TER, whereas LMW-HA induced biphasic TER changes ultimately resulting in EC barrier disruption. HMW-HA induced the association of the CD44s isoform with, and AKT-mediated phosphorylation of, the barrierpromoting sphingosine 1-phosphate receptor (S1P 1 ) within caveolin-enriched lipid raft microdomains, whereas LMW-HA induced brief CD44s association with S1P 1 followed by sustained association of the CD44v10 isoform with, and Src and ROCK 1/2-mediated phosphorylation of, the barrier-disrupting S1P 3 receptor. HA-induced EC cytoskeletal reorganization and TER alterations were abolished by either disruption of lipid raft formation, CD44 blocking antibody or siRNA-mediated reductions in expression of CD44 isoforms. Silencing S1P 1 , AKT1, or Rac1 blocked the barrier enhancing effects of HA whereas silencing S1P 3 , Src, ROCK1/2, or RhoA blocked the barrier disruption induced by LMW-HA. In summary, HA regulates EC barrier function through novel differential CD44 isoform interaction with S1P receptors, S1P receptor transactivation, and RhoA/Rac1 signaling to the EC cytoskeleton.

In this study we have examined CD44 (a hyaluronan (HA) receptor) interaction with a RhoA-specific guanine nucleotide exchange factor (p115RhoGEF) in human metastatic breast tumor cells (MDA-MB-231 cell line). Immunoprecipitation and immunoblot analyses indicate that both CD44 and p115RhoGEF are expressed in MDA-MB-231 cells and that these two proteins are physically associated as a complex in vivo. The binding of HA to MDA-MB-231 cells stimulates p115RhoGEF-mediated RhoA signaling and Rho kinase (ROK) activity, which, in turn, increases serine/threonine phosphorylation of the adaptor protein, Gab-1 (Grb2-associated binder-1). Phosphorylated Gab-1 promotes PI 3-kinase recruitment to CD44v3. Subsequently, PI 3-kinase is activated (in particular, ␣, ␤, ␥ forms but not the ␦ form of the p110 catalytic subunit), AKT signaling occurs, the cytokine (macrophage-colony stimulating factor (M-CSF)) is produced, and tumor cell-specific phenotypes (e.g. tumor cell growth, survival and invasion) are up-regulated. Our results also demonstrate that HA/CD44-mediated oncogenic events (e.g. AKT activation, M-CSF production and breast tumor cell-specific phenotypes) can be effectively blocked by a PI 3-kinase inhibitor (LY294002). Finally, we have found that overexpression of a dominant-negative form of ROK (by transfection of MBA-MD-231 cells with the Rho-binding domain cDNA of ROK) not only inhibits HA/CD44-mediated RhoA-ROK activation and Gab-1 phosphorylation but also downregulates oncogenic signaling events (e.g. Gab-1⅐PI 3-kinase-CD44v3 association, PI 3-kinase-mediated AKT activation, and M-CSF production) and tumor cell behaviors (e.g. cell growth, survival, and invasion). Taken together, these findings strongly suggest that CD44 interaction with p115RhoGEF and ROK plays a pivotal role in promoting Gab-1 phosphorylation leading to Gab-1⅐PI 3-kinase membrane localization, AKT signaling, and cytokine (M-CSF) production during HAmediated breast cancer progression.

Background The possibility that mu opioid agonists can influence cancer recurrence is a subject of recent interest. Epidemiologic studies suggested that there were differences in cancer recurrence in breast and prostate cancer contingent on anesthetic regimens. In this study, we identify a possible mechanism for these epidemiologic findings based on mu opioid receptor (MOR) regulation of Lewis lung carcinoma (LLC) tumorigenicity in cell and animal models. Methods We utilized human lung tissue and human non-small cell lung cancer (NSCLC) cell lines and evaluated MOR expression using immunoblot and immunohistochemical analysis. LLC cells were treated with the peripheral opioid antagonist methylnalnaltrexone (MNTX) or MOR shRNA and evaluated for proliferation, invasion and soft agar colony formation in vitro and primary tumor growth and lung metastasis in C57BL/6 and MOR knockout mice using Visen FMT imaging and immunohistochemical analysis. Results We provide several lines of evidence that the MOR may be a potential target for lung cancer, a disease with high mortality and few treatment options. We first observed that there is ~5 to 10 fold increase in MOR expression in lung samples from patients with NSCLC and in several human NSCLC cell lines. The MOR agonists morphine and DAMGO increased in vitro LLC cell growth. Treatment with MNTX or silencing MOR expression inhibited LLC invasion and anchorage-independent growth by 50–80%. Injection of MOR silenced LLC lead to a ~65% reduction in mouse lung metastasis. In addition, MOR knockout mice do not develop significant tumors when injected with LLC as compared to wildtype controls. Finally, continuous infusion of the peripheral opioid antagonist methylnaltrexone attenuates primary LLC tumor growth and reduces lung metastasis. Conclusions Taken together, our data suggests a possible direct effect of opiates on lung cancer progression, and provides a plausible explanation for the epidemiologic findings. Our observations further suggest a possible therapeutic role for opioid antagonists.

In this study we initially examined the interaction between CD44v3 (a hyaluronan (HA) receptor) and Vav2 (a guanine nucleotide exchange factor) in human ovarian tumor cells (SK-OV-3.ipl cell line). Immunological data indicate that both CD44v3 and Vav2 are expressed in SK-OV-3.ipl cells and that these two proteins are physically linked as a complex in vivo. By using recombinant fragments of Vav2 and in vitro binding assays, we have detected a specific binding interaction between the SH3-SH2-SH3 domain of Vav2 and the cytoplasmic domain of CD44. In addition, we have observed that the binding of HA to CD44v3 activates Vav2-mediated Rac1 signaling leading to ovarian tumor cell migration. Further analyses indicate that the adaptor molecule, growth factor receptor-bound protein 2 (Grb2) that is bound to p185 HER2 (an oncogene product), is also associated with the CD44v3-Vav2 complex. HA binding to SK-OV-3.ipl cells promotes recruitment of both Grb2 and p185 HER2 to the CD44v3-Vav2 complex leading to Ras activation and ovarian tumor cell growth. In order to determine the role of Grb2 in CD44v3 signaling, we have transfected SK-OV-3.ipl cells with Grb2 mutant cDNAs (e.g. ⌬N-Grb2 that has a deletion in the amino-terminal SH3 domain or ⌬C-Grb2 that has a deletion in the carboxyl-terminal SH3 domain). Our results clearly indicate that the SH3 domain deletion mutants of Grb2 (i.e. the ⌬N-Grb2 (and to a lesser extent the ⌬C-Grb2) mutant) not only block their association with p185 HER2 but also significantly impair their binding to the CD44v3-Vav2 complex and inhibit HA/CD44v3-induced ovarian tumor cell behaviors. Taken together, these findings strongly suggest that the interaction of CD44v3-Vav2 with Grb2-p185 HER2 plays an important role in the coactivation of both Rac1 and Ras signaling that is required for HA-mediated human ovarian tumor progression.The cell adhesion molecule, CD44, is a product of a single gene that undergoes alternative splicing of 12 possible exons to generate variant isoforms (1). Nucleotide sequence analyses reveal that the CD44 isoforms are variants of the standard form, CD44s (1). CD44s (molecular mass Ϸ85 kDa) is the most common isoform of CD44 found in many cell types including human ovarian carcinoma cells (2). CD44 can be further modified by extensive N-and O-glycosylations and glycosaminoglycan additions (3-5). Apparently, both post-translational modifications and/or alternative splicing within the CD44 structure determine the functional outcome of this molecule. CD44 is a transmembrane glycoprotein that is one of the major hyaluronan (HA) 1 receptors (6). CD44 binds to extracellular matrix components (i.e. HA) at its amino terminus of the extracellular domain (7,8). CD44 also contains specific binding sites for the cytoskeleton (9 -14) and various signaling molecules (15-19) within the 70-amino acid carboxyl terminus in the cytoplasmic domain.Both CD44 and HA appear to be overexpressed at sites of tumor attachment and are known to be involved in cell aggregation, proliferation, migration, and...

In this study we have examined the interaction of CD44 (a major hyaluronan (HA) receptor) with a RhoA-specific guanine nucleotide exchange factor (leukemia-associated RhoGEF (LARG)) in human head and neck squamous carcinoma cells (HNSCC- Human head and neck squamous cell carcinoma (HNSCC) 2 is a very malignant cancer associated with major morbidity and mortality. This deadly disease includes cancers of the lips, oral cavity, pharynx, hypopharynx, larynx, nose, nasal, sinuses, neck, ears, and salivary glands (1-6). Approximately 90% of HNSCCs are very aggressive and rapidly invade the surrounding tissues. Because little is known about the molecular basis underlying the progression of HNSCC to the invasive phenotype, it is very difficult to predict individual tumor aggressiveness and to design effective treatment plans. Thus, there is currently a great need to clarify those aspects of tumor formation underlying the clinical behavior of HNSCC.HSCA number of studies have aimed at identifying the specific molecules expressed in HNSCC that correlate with invasive behavior. Among such candidates are hyaluronan (HA) (7, 8) and its major cell surface receptor, CD44 (9). HA is a major component of the extracellular matrix component and is significantly enriched in many types of tumors (7,8). HA binds to its specific cell surface receptor, CD44, a multifunctional transmembrane glycoprotein expressed in many cells and tissues including HNSCC cells and carcinoma tissues (10 -14). CD44 is often expressed as a variety of variant isoforms, generated by an alternative splicing mechanism (15). The expression of certain CD44 variant (CD44v) isoforms is known to be associated with head and neck cancer progression (10 -14).The external portion of CD44 binds HA (9), whereas the intracellular domain interacts with specific signaling molecules such as RhoA-activated Rho kinase (16) and Rho/Rac1-specific guanine nucleotide exchange factors (p115RhoGEF (17), Tiam1 (18), and Vav2 (19)), c-Src kinase (20), protein kinase N␥ (21), IQGAP1 (22), p185 HER2 (23), and transforming growth factor-␤ receptors (24)). CD44 also binds directly to cytoskeletal proteins such as ankyrin and ezrin, radixin, and moesin (25-28). The interaction of CD44 with the cytoskeleton and various signaling molecules plays a pivotal role in promoting metastatic-specific tumor phenotypes such as matrix metalloproteinase (MMP)-mediated matrix degradation, tumor cell growth, migration, and invasion (10, 16 -28 2 The abbreviations used are: HNSCC, human head and neck squamous cell carcinoma; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; HA, hyaluronan; MMP, matrix metalloproteinase; EGFR, epidermal growth factor (EGF) receptor; GEF, guanine nucleotide exchange factor; LARG, leukemia-associated RhoGEF; DH, dbl homology; PH, pleckstrin homology; PLC, phospholipase C; IP 3 , inositol 1,4,5-triphosphate; CaMKII, Ca 2ϩ /calmodulin-dependent kinase II; GST, glutathione S-transferase; FITC, fluorescein isothiocyanate; BSA, bovine serum alb...