CD44 signaling through focal adhesion kinase and its anti-apoptotic effect

Adhesion molecules can initiate intracellular signaling. Engagement of CD44 either by its natural ligand hyaluronan or a specific antibody on a cell line induced tyrosine phosphorylation and activation of focal adhesion kinase (FAK), which then associated with phosphatidylinositol 3-kinase (PI3K) and activated mitogen-activated protein kinase at its downstream. However, the introduction of dominant negative Rho into the cells inhibited the CD44-stimulated FAK phosphorylation. Cells expressing CD44 were significantly resistant to etoposide-induced apoptosis. This anti-apoptotic effect was cancelled by the inhibition of either Rho, FAK or PI3K. These results may indicate a signaling pathway from CD44 to mediate the resistance against drug-induced apoptosis in cancer cells.     

Proneural transcription factors regulate different steps of cortical neuron migration through Rnd-mediated inhibition of RhoA signaling

Little is known of the intracellular machinery that controls the motility of newborn neurons. We have previously shown that the proneural protein Neurog2 promotes the migration of nascent cortical neurons by inducing the expression of the atypical Rho GTPase Rnd2. Here, we show that another proneural factor, Ascl1, promotes neuronal migration in the cortex through direct regulation of a second Rnd family member, Rnd3. Both Rnd2 and Rnd3 promote neuronal migration by inhibiting RhoA signaling, but they control distinct steps of the migratory process, multipolar to bipolar transition in the intermediate zone and locomotion in the cortical plate, respectively. Interestingly, these divergent functions directly result from the distinct subcellular distributions of the two Rnd proteins. Because Rnd proteins also regulate progenitor divisions and neurite outgrowth, we propose that proneural factors, through spatiotemporal regulation of Rnd proteins, integrate the process of neuronal migration with other events in the neurogenic program.     

Galectin-9 induces osteoblast differentiation through the CD44/Smad signaling pathway

Galectin-9 is a β-galactoside-binding lectin expressed in various tissues. It binds various glycoconjugates and modulates a variety of biological functions in various cell types. Although galectin-9 is expressed in bone, its function in human osteoblasts remains unclear. We demonstrate that galectin-9 induces osteoblast differentiation through the CD44/Smad signaling pathway in the absence of bone morphogenetic proteins (BMPs). Galectin-9 increases alkaline phosphatase activities in human osteoblasts and induces the phosphorylation of Smad1/5/8 and translocation of Smad4 to the nucleus in the absence of BMPs. Galectin-9 also induces binding of Smad4 to the Id1 promoter and increases its activity. Anti-CD44 antibody inhibits Smad1/5/8 phosphorylation by galectin-9. Galectin-9 binds to CD44 and induces the formation of a CD44/BMP receptor complex. Because Smad1 is phosphorylated by BMP receptors, we propose that formation of the CD44/BMP receptor complex induced by galectin-9 may provide a trigger for the activation of Smads.     

Transgelin-2 interacts with CD44 to regulate Notch1 signaling pathway and participates in colorectal cancer proliferation and migration

Journal of Physiology and Biochemistry - The abnormal expression of transgelin-2 (TAGLN2) is related to tumor occurrence and progression. However, the underlying molecular mechanism of TAGLN2 in...     

Rheostatic signaling by CD44 and hyaluronan

Cellular function and adaptive behavior is often driven by signals generated in response to the local tissue microenvironment. Cell surface receptors that detect changes in extracellular matrix composition and modifications to extracellular matrix components, are ideally positioned to provide highly responsive sensors of changes in the microenvironment and mediate changes in cellular function required to maintain tissue integrity. Receptors can act as “on/off” switches, but ligand/receptor complexes that provide “rheostatic” control may be more sensitive, provide a more rapid mechanism of control and allow for fine-tuning of cellular responses to the microenvironment. Herein, we review evidence that transitions in the physiochemical properties of the extracellular glycosaminoglycan hyaluronan and in the function of its major receptor, CD44, differentially regulate ERK and Rac signal transduction pathways to provide critical rheostatic control of mesenchymal cell proliferation.     

Thrombospondin induces RhoA inactivation through FAK-dependent signaling to stimulate focal adhesion disassembly

Cells utilize dynamic interactions with the extracellular matrix to adapt to changing environmental conditions. Thrombospondin 1 (TSP1) induces focal adhesion disassembly and cell migration through a sequence (hep I) in its heparin-binding domain signaling through the calreticulin-low density lipoprotein receptor-related protein receptor complex. This involves the Galphai-dependent activation of ERK and phosphoinositide (PI) 3-kinase, both of which are required for focal adhesion disassembly. Focal adhesion kinase (FAK) regulates adhesion dynamics, acting in part by modulating RhoA activity, and FAK is implicated in ERK and PI 3-kinase activation. In this work, we sought to determine the role of FAK in TSP1-induced focal adhesion disassembly. TSP1/hep I does not stimulate focal adhesion disassembly in FAK knockout fibroblasts, whereas re-expressing FAK rescues responsiveness. Inhibiting FAK signaling through FRNK or FAK Y397F expression in endothelial cells also abrogates this response. TSP1/hep I stimulates a transient increase in FAK phosphorylation that requires calreticulin and Galphai, but not ERK or PI 3-kinase. Hep I does not activate ERK or PI 3-kinase in FAK knockout fibroblasts, suggesting activation occurs downstream of FAK. TSP1/hep I stimulates RhoA inactivation with kinetics corresponding to focal adhesion disassembly in a FAK, ERK, and PI 3-kinase-dependent manner. Furthermore, hep I does not stimulate focal adhesion disassembly in cells expressing constitutively active RhoA, suggesting that RhoA inactivation is required for this response. This is the first work to illustrate a connection between FAK phosphorylation in response to a soluble factor and RhoA inactivation, as well as the first report of PI 3-kinase and ERK in FAK regulation of RhoA activity.     

CD2 and CD3 associate independently with CD5 and differentially regulate signaling through CD5 in Jurkat T cells.

In T lymphocytes, the CD2 and CD5 glycoproteins are believed to be involved in the regulation of signals elicited by the TCR/CD3 complex. Here we show that CD2 and CD3 independently associate with CD5 in human PBMC and Jurkat cells. CD5 coprecipitates with CD2 in CD3-deficient cells and, conversely, coprecipitates with CD3 in cells devoid of CD2. In unstimulated CD2+ CD3+ Jurkat cells, CD5 associates equivalently with CD2 and CD3 and is as efficiently phosphorylated in CD2 as in CD3 immune complexes. However, upon activation the involvement of CD5 is the opposite in the CD2 and CD3 pathways. CD5 becomes rapidly tyrosine phosphorylated after CD3 stimulation, but is dephosphorylated upon CD2 cross-linking. These opposing effects correlate with the decrease in the activity of the SH2 domain-containing protein phosphatase 1 (SHP-1) following CD3 activation vs an enhanced activity of the phosphatase after CD2 triggering. The failure of CD5 to become phosphorylated on tyrosine residues in the CD2 pathway has no parallel with the lack of use of zeta-chains in CD2 signaling; contrasting with comparable levels of association of CD2 or CD3 with CD5, zeta associates with CD2 only residually and is nevertheless slightly phosphorylated after CD2 stimulation. The modulation of CD5 phosphorylation may thus represent a level of regulation controlled by CD2 in signal transduction mechanisms in human T lymphocytes.     

Cytokines regulate the affinity of soluble CD44 for hyaluronan

DNA enzymes are RNA-cleaving single-stranded DNA molecules. We designed DNA enzymes targeting the PB2 mRNA translation initiation (AUG) region of the influenza A virus (A/PR/8/34). The modified DNA enzymes have one or two N3′-P5′ phosphoramidate bonds at both the 3′- and 5′-termini of the oligonucleotides, which significantly enhanced their nuclease resistance. These modified DNA enzymes had the same cleavage activity as the unmodified DNA enzymes, determined by kinetic analyses, and reduced influenza A virus replication by more than 99%, determined by plaque formation. These DNA enzymes are highly specific; their protective effect was not observed in influenza B virus (B/Ibaraki)-infected Madin–Darby canine kidney cells.     

Growth and differentiation regulate CD44 expression on human keratinocytes

Summary Several members of the CD44 family of hyaluronan receptors are expressed on keratinocytes. To identify factors that might be important in regulating CD44 expression, we studied CD44 expression on keratinocytes growing in vitro under a variety of conditions and on cells isolated directly from epidermis. Using Western immunoblots and metabolic labeling, we showed that the pattern of CD44 proteins expressed by keratinocytes was strongly influenced by growth and differentiation. Many protein forms of CD44 are expressed on proliferating keratinocytes in preconfluent cultures, whereas only a few forms are expressed on differentiated cells and in confluent cultures. In preconfluent monolayers, at least four splice variants were identified, including epican, CD44H, CD44E, and a 180-kDa variant. In differentiated cells or in confluent cultures, by contrast, only epican and the 180-kDa protein variant were found. Synthesis of all variants is strongly downregulated when keratinocytes become confluent or when they differentiate. Epican is the predominant form of CD44 on keratinocytes under all conditions and is expressed as a heparan, chondroitin, or keratan sulfate proteoglycan. Preconfluent basal keratinocytes, but not confluent or differentiated keratinocytes, also express chondroitin sulfate proteoglycan forms of CD44E and of the 180-kDa core protein. The modal size of the epican expressed on differentiated keratinocytes is smaller than the size of the epican expressed on basal keratinocytes. Thus, cell confluence and differentiation regulate several aspects of CD44 expression on keratinocytes, suggesting nuances in function for the different protein forms.     

PKC-1 acts with the ERK MAPK signaling pathway to regulate Caenorhabditis elegans mechanosensory response

In most animals, multiple genes encode protein kinase C (PKC) proteins. Pharmacological studies have revealed numerous roles for this protein family, yet the in vivo roles of specific PKC proteins and the functional targets of PKC activation are poorly understood. We find that in Caenorhabditis elegans, two PKC genes, pkc-1 and tpa-1, are required for mechanosensory response; the role of the nPKCε/η ortholog, pkc-1, was examined in detail. pkc-1 function is required for response to nose touch in adult C. elegans and pkc-1 likely acts in the interneurons that regulate locomotion which are direct synaptic targets of mechanosensory neurons. Previous studies have suggested numerous possible targets of pkc-1; our analysis indicates that pkc-1 may act via the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway. We find that ERK/MAPK pathway function is required for mechanosensory response in C. elegans and that at least one component of this pathway, lin-45 Raf, acts in interneurons of the mechanosensory circuit. Genetic analysis indicates that lin-45 and pkc-1 act together to regulate nose touch response. Thus, these results functionally link two conserved signaling pathways in adult C. elegans neurons and define distinct roles for PKC genes in vivo.     

CD44 enhances neuregulin signaling by Schwann cells

We describe a key role for the CD44 transmembrane glycoprotein in Schwann cell-neuron interactions. CD44 proteins have been implicated in cell adhesion and in the presentation of growth factors to high affinity receptors. We observed high CD44 expression in early rat neonatal nerves at times when Schwann cells proliferate but low expression in adult nerves, where CD44 was found in some nonmyelinating Schwann cells and to varying extents in some myelinating fibers. CD44 constitutively associated with erbB2 and erbB3, receptor tyrosine kinases that heterodimerize and signal in Schwann cells in response to neuregulins. Moreover, CD44 significantly enhanced neuregulin-induced erbB2 phosphorylation and erbB2-erbB3 heterodimerization. Reduction of CD44 expression in vitro resulted in loss of Schwann cell-neurite adhesion and Schwann cell apoptosis. CD44 is therefore crucial for maintaining neuron-Schwann cell interactions at least partly by facilitating neuregulin-induced erbB2-erbB3 activation.     

CD44 engagement promotes matrix-derived survival through the CD44-SRC-integrin axis in lipid rafts

CD44 is present in detergent-resistant, cholesterol-rich microdomains, called lipid rafts, in many types of cells. However, the functional significance of CD44 in lipid rafts is still unknown. We have previously demonstrated that osteopontin-mediated engagement of CD44 spliced variant isoforms promotes an extracellular matrix-derived survival signal through integrin activation. By using a series of CD44 mutants and pharmacological inhibitors selectively targeted to various cellular pathways, we show in this study that engagement of CD44 induces lipid raft coalescence to facilitate a CD44-Src-integrin signaling axis in lipid rafts, leading to increased matrix-derived survival. Palmitoylation of the membrane-proximal cysteine residues and carboxyl-terminal linkage to the actin cytoskeleton both contribute to raft targeting of CD44. The enrichment of integrin β1 in lipid rafts is tightly coupled to CD44 ligation-elicited lipid raft reorganization and associated with temporally delayed endocytosis. Through the interaction with the CD44 carboxyl-terminal ankyrin domain, Src is cotranslocated to lipid rafts, where it induces integrin activation via an inside-out mechanism. Collectively, this study demonstrates an important role of the dynamic raft reorganization induced by CD44 clustering in eliciting the matrix-derived survival signal.     

Trans-acting factors regulate the expression of CD44 splice variants.

Variant isoforms of the cell surface glycoprotein CD44 (CD44v) are expressed during development, in selected adult tissues and in certain metastatic tumor cells. CD44v differ from the standard isoform (CD44s) by up to ten additional exon sequences included by alternative splicing. By cell fusion experiments, we have obtained evidence for the existence of cell-type specific trans-acting factors recruiting CD44 variant exon sequences. Stable cell hybrids of CD44s and CD44v expressing cells indicated a dominant mechanism for variant-exon inclusion. In transient interspecies heterokaryons of human keratinocytes and rat fibroblasts, the ability of the keratinocytes to include all variant exon sequences in CD44 was conferred completely on the rat fibroblast nucleus. Fusions of cells with complex CD44 splice patterns do not permit interpretation of splice control by the relative abundance of a single trans-acting factor, but rather by (a) positively acting factor(s) recruiting variant exon sequences in the 3' to 5' direction and additional factors selecting individual exons. Since the pancreatic carcinoma cell line BSp73ASML (in contrast to the cervix carcinoma cell lines SiHa and ME180) could not transfer its specific splice pattern in cell fusions, we conclude that in some tumors, splicing is also controlled by mutation of cis-acting recognition sites.     

RhoA co-ordinates with heterotrimeric G proteins to regulate efficacy

Heterotrimeric G proteins have a critical role in mediating signal transduction by ligand-stimulated GPCRs. While activation of heterotrimeric G proteins is known to proceed via the G protein guanine nucleotide cycle, there is much uncertainty regarding the process that determines efficacy, the extent of response across signaling pathways. Gα_GTP can interact with multiple binding partners, including several effectors and regulators. Cross-talk by other receptor-signaling pathways can alter the response. It remains unclear whether G protein efficacy is regulated. This lack of clarity impairs our ability to predict and manipulate the pharmacological behavior of activated G proteins. This review will discuss emerging evidence that implicates monomeric RhoA in the process that regulates G_q efficacy.     

JNK phosphorylates Yes-associated protein (YAP) to regulate apoptosis

Yes-associated protein (YAP) regulates DNA damage and chemosensitivity, as well as functioning as a pro-growth, cell size regulator. For both of its roles, regulation by phosphorylation is crucial. We undertook an in vitro screen to identify novel YAP kinases to discover new signaling pathways to better understand YAP''s function. We identified JNK1 and JNK2 as robust YAP kinases, as well as mapped multiple sites of phosphorylation. Using inhibitors and siRNA, we showed that JNK specifically phosphorylates endogenous YAP in a number of cell types. We show that YAP protects keratinocytes from UV irradiation but promotes UV-induced apoptosis in a squamous cell carcinoma. We defined the mechanism for this dual role to be YAP''s ability to bind and stabilize the pro-proliferative ΔNp63α isoform in a JNK-dependent manner. Our report indicates that an evaluation of the expression of the different isoforms of p63 and p73 is crucial in determining YAP''s function.