Hyaluronan-CD44 interaction with leukemia-associated RhoGEF and epidermal growth factor receptor promotes Rho/Ras co-activation, phospholipase C epsilon-Ca2+ signaling, and cytoskeleton modification in head and neck squamous cell carcinoma cells.
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-HSC-3 cell line). Immunoprecipitation and immunoblot analyses indicate that CD44 and the LARG protein are expressed in HSC-3 cells and that these two proteins are physically associated as a complex. HA-CD44 binding induces LARG-specific RhoA signaling and phospholipase C epsilon (PLC epsilon) activity. In particular, the activation of RhoA-PLC epsilon by HA stimulates inositol 1,4,5-triphosphate production, intracellular Ca2+ mobilization, and the up-regulation of Ca2+/calmodulin-dependent kinase II (CaMKII), leading to phosphorylation of the cytoskeletal protein, filamin. The phosphorylation of filamin reduces its interaction with filamentous actin, promoting tumor cell migration. The CD44-LARG complex also interacts with the EGF receptor (EGFR). Most importantly, the binding of HA to the CD44-LARG-EGFR complex activates the EGFR receptor kinase, which in turn promotes Ras-mediated stimulation of a downstream kinase cascade including the Raf-1 and ERK pathways leading to HNSCC cell growth. Using a recombinant fragment of LARG (the LARG-PDZ domain) and a binding assay, we have determined that the LARG-PDZ domain serves as a direct linker between CD44 and EGFR. Transfection of the HSC-3 cells with LARG-PDZcDNA significantly reduces LARG association with CD44 and EGFR. Overexpression of the LARG-PDZ domain also functions as a dominant-negative mutant (similar to the PLC/Ca2+-calmodulin-dependent kinase II (CaMKII) and EGFR/MAPK inhibitor effects) to block HA/CD44-mediated signaling events (e.g. EGFR kinase activation, Ras/RhoA co-activation, Raf-ERK signaling, PLC epsilon-mediated inositol 1,4,5-triphosphate production, intracellular Ca2+ mobilization, CaMKII activity, filamin phosphorylation, and filamin-actin binding) and to abrogate tumor cell growth/migration. Taken together, our findings suggest that CD44 interaction with LARG and EGFR plays a pivotal role in Rho/Ras co-activation, PLC epsilon-Ca2+ signaling, and Raf/ERK up-regulation required for CaMKII-mediated cytoskeleton function and in head and neck squamous cell carcinoma progression.
Department of Medicine, University of California at San Francisco and Endocrine Unit (111N), Veterans Affairs Medical Center, San Francisco, California 94121, USA. lillyb@itsa.ucsf.edu
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D002294:Carcinoma, Squamous Cell / Q000378:metabolism*; D045744:Cell Line, Tumor; D003599:Cytoskeleton / Q000378:metabolism; D066246:ErbB Receptors / Q000737:chemistry* / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry* / Q000378:metabolism; D006258:Head and Neck Neoplasms / Q000378:metabolism*; D006801:Humans; D018960:Hyaluronan Receptors / Q000737:chemistry* / Q000378:metabolism; D006820:Hyaluronic Acid / Q000737:chemistry* / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D054801:Phosphoinositide Phospholipase C; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D064067:Rho Guanine Nucleotide Exchange Factors; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000378:metabolism; D018631:ras Proteins / Q000378:metabolism; D054460:rho-Associated Kinases
C577222:ARHGEF12 protein, human; D020662:Guanine Nucleotide Exchange Factors; D018960:Hyaluronan Receptors; D047908:Intracellular Signaling Peptides and Proteins; D064067:Rho Guanine Nucleotide Exchange Factors; D006820:Hyaluronic Acid; D066246:ErbB Receptors; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; C423320:phospholipase C epsilon; D018631:ras Proteins