Downregulation of protein kinase C gamma reduces epithelial property and enhances malignant phenotypes in colorectal cancer cells.

Loss of epithelial integrity is associated with colorectal cancer (CRC) aggressiveness. Protein kinase C (PKC) is frequently implicated in human cancers, but the role of PKCγ in CRC remains poorly understood. Here, we show that PKCγ, a conventional PKC, is expressed in normal colonic epithelium, but this is lower in dedifferentiated CRC. PKCγ expression was downregulated by SNAI1 overexpression, and low PKCγ expression was associated with poor prognosis in patients with CRC. Transient or stable knockdown of PKCγ reduced E-cadherin expression in CRC cells. PKCγ knockdown enhanced proliferation, anchorage-independent cell growth, resistance to anti-cancer drugs, and in vivo tumor growth of DLD-1 cells. We have also identified phosphorylation substrates for PKCγ. Among them, ARHGEF18, a RhoA activator that stabilizes cell-cell junctions, was phosphorylated and stabilized by PKCγ. Thus, these results suggest that the downregulation of PKCγ decreases the epithelial property of CRC cells and enhances its malignant phenotypes.

Patulin and LL-Z1640-2 induce apoptosis of cancer cells by decreasing endogenous protein levels of Zic family member 5.

Zic family member 5 (ZIC5) is a transcription factor that promotes the survival of several cancer cell types. As ZIC5 is expressed at minimal levels in normal human adult tissues, it is a potential therapeutic target. In this study, we screened a chemical library containing 3398 compounds that includes pre-existing drugs and compounds with known effects to identify ZIC5 inhibitors. In the first screening, 18 hit compounds decreased GFP intensity in melanoma A375 cells overexpressing GFP-tagged ZIC5. In the second screening, five compounds that attenuated ZIC5 protein levels in A375 cells were identified. Among them, LL-Z1640-2 and patulin selectively induced apoptosis in melanoma cells expressing ZIC5, while only inducing very low levels of apoptosis in normal human melanocytes, which have no detectable ZIC5 expression. LL-Z1640-2 and patulin also induced apoptosis in BRAF inhibitor-resistant melanoma, pancreatic cancer, cholangiocarcinoma and colorectal cancer cells. LL-Z1640-2- and patulin-mediated suppression of melanoma proliferation were rescued by ZIC5 overexpression. These results suggest that LL-Z1640-2 and patulin are promising compounds that decrease ZIC5 expression to induce apoptosis in cancer cells.

Zic family member 5 promotes survival in human pancreatic cancer and cholangiocarcinoma cells.

Pancreatic ductal adenocarcinoma (PDAC) and cholangiocarcinoma (CCA) are malignant tumors with poor prognosis because of the limited effectiveness of traditional chemotherapy and few effective molecular therapeutic agents. Here, we determined the essential roles of Zic family member 5 (ZIC5) in the survival of PDAC and CCA cells. The results showed that ZIC5 is strongly expressed in PDAC and CCA tissues, while ZIC5 expression is barely observed in most normal human adult tissues. Furthermore, ZIC5 expression is related to poor prognosis of patients with PDAC. ZIC5 knockdown via small interfering RNA decreased the phosphorylation of signal transducer and activator of transcription 3 (STAT3), a protein that is associated with PDAC and CCA aggressiveness. However, ZIC5 knockdown induced cell death regardless of STAT3 activation, which is promoted by interleukin (IL) -6, a factor associated with inflammation. Furthermore, knockdown of ZIC5 in PDAC and CCA cells additively or synergistically induced apoptosis with the anti-cancer drug gemcitabine. Thus, ZIC5 constitutes a potential therapeutic target for the treatment of PDAC and CCA.

Immunoregulatory Monocyte Subset Promotes Metastasis Associated With Therapeutic Intervention for Primary Tumor.

Systemic and local inflammation associated with therapeutic intervention of primary tumor occasionally promotes metastatic recurrence in mouse and human. However, it remains unclear what types of immune cells are involved in this process. Here, we found that the tissue-repair-promoting Ym1+Ly6Chi monocyte subset expanded as a result of systemic and local inflammation induced by intravenous injection of lipopolysaccharide or resection of primary tumor and promoted lung metastasis originating from circulating tumor cells (CTCs). Deletion of this subset suppressed metastasis induced by the inflammation. Furthermore, transfer of Ym1+Ly6Chi monocytes into naïve mice promoted lung metastasis in the mice. Ym1+Ly6Chi monocytes highly expressed matrix metalloproteinase-9 (MMP-9) and CXCR4. MMP-9 inhibitor and CXCR4 antagonist decreased Ym1+Ly6Chi-monocyte-promoted lung metastasis. These findings indicate that Ym1+Ly6Chi monocytes are therapeutic target cells for metastasis originating from CTCs associated with systemic and local inflammation. In addition, these findings provide a novel predictive cellular biomarker for metastatic recurrence after intervention for primary tumor.

Phosphatidylinositol 4,5-bisphosphate is localized in the plasma membrane outer leaflet and regulates cell adhesion and motility.

Phospholipids are distributed asymmetrically in the plasma membrane (PM) of mammalian cells. Phosphatidylinositol (PI) and its phosphorylated forms are primarily located in the inner leaflet of the PM. Among them, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a well-known substrate for phospholipase C (PLC) or phosphoinositide-3 kinase, and is also a regulator for the actin cytoskeleton or ion channels. Although functions of PI(4,5)P2 in the inner leaflet are well characterized, those in the outer leaflet are poorly understood. Here, PI(4,5)P2 was detected in the cell surface of non-permeabilized cells by anti-PI(4,5)P2 antibodies and the pleckstrin-homology (PH) domain of PLCδ1 that specifically binds PI(4,5)P2. Cell surface PI(4,5)P2 signal was universally detected in various cell lines and freshly isolated mouse bone marrow cells and showed a punctate pattern in a cholesterol, sphingomyelin, and actin polymerization-dependent manner. Furthermore, blocking cell surface PI(4,5)P2 by the addition of anti-PI(4,5)P2 antibody or the PH domain of PLCδ1 inhibited cell attachment, spreading, and migration. Taken together, these results indicate a unique localization of PI(4,5)P2 in the outer leaflet that may have a crucial role in cell attachment, spreading, and migration.

Cell surface CD63 increased by up-regulated polylactosamine modification sensitizes human melanoma cells to the BRAF inhibitor PLX4032.

The BRAF inhibitor PLX4032 is effective in treating BRAF-mutated melanoma; however, because drug resistance develops in most cases, it is critical to develop a new strategy for inhibiting drug-resistant melanoma growth. The melanoma-associated membrane glycoprotein CD63 is involved in cell proliferation and metastasis. Here, we found that cell surface CD63 suppresses the proliferation of human melanoma cells and PLX4032-resistant cells. Endogenous CD63 protein levels were negatively correlated with PLX4032 resistance of human melanoma cell lines. CD63 overexpression in these cells, in which endogenous CD63 levels are low, suppressed cell proliferation under PLX4032 treatment. The cell surface levels and average molecular mass of CD63 were increased with PLX4032 treatment because of the up-regulated polylactosamine modification caused by induced ß1,3- N-acetylglucosaminyltransferase 2 expression, which is involved in polylactosamine synthesis. Forced cell surface localization of CD63 led to reduced melanoma cell proliferation without PLX4032 treatment. CD63 overexpression in PLX4032-resistant cells, in which CD63 levels were lower and cell surface polylactosamine levels were higher than those in parental cells, effectively suppressed proliferation. Our study shows the potential of CD63 to sensitize melanoma cells to PLX4032 and to reduce the proliferation of PLX4032-resistant cells.-Kudo, K., Yoneda, A., Sakiyama, D., Kojima, K., Miyaji, T., Yamazaki, M., Yaita, S., Hyodo, T., Satow, R., Fukami, K. Cell surface CD63 increased by up-regulated polylactosamine modification sensitizes human melanoma cells to the BRAF inhibitor PLX4032.

Phospholipase Cδ1 regulates p38 MAPK activity and skin barrier integrity.

Keratinocytes undergo a unique type of programmed cell death known as cornification, which leads to the formation of the stratum corneum (SC), the main physical barrier of the epidermis. A defective epidermal barrier is a hallmark of the two most common inflammatory skin disorders, psoriasis, and atopic dermatitis. However, the detailed molecular mechanisms of skin barrier formation are not yet fully understood. Here, we showed that downregulation of phospholipase C (PLC) δ1, a Ca2+-mobilizing and phosphoinositide-metabolizing enzyme abundantly expressed in the epidermis, impairs the barrier functions of the SC. PLCδ1 downregulation also impairs localization of tight junction proteins. Loss of PLCδ1 leads to a decrease in intracellular Ca2+ concentrations and nuclear factor of activated T cells activity, along with hyperactivation of p38 mitogen-activated protein kinase (MAPK) and inactivation of RhoA. Treatment with a p38 MAPK inhibitor reverses the barrier defects caused by PLCδ1 downregulation. Interestingly, this treatment also attenuates psoriasis-like skin inflammation in imiquimod-treated mice. These findings demonstrate that PLCδ1 is essential for epidermal barrier integrity. This study also suggests a possible link between PLCδ1 downregulation, p38 MAPK hyperactivation, and barrier defects in psoriasis-like skin inflammation.

The Phosphorylation and Distribution of Cortactin Downstream of Integrin α9ß1 Affects Cancer Cell Behaviour.

Integrins, a family of heterodimeric adhesion receptors are implicated in cell migration, development and cancer progression. They can adopt conformations that reflect their activation states and thereby impact adhesion strength and migration. Integrins in an intermediate activation state may be optimal for migration and we have shown previously that fully activated integrin α9ß1 corresponds with less migratory behaviour in melanoma cells. Here, we aimed to identify components associated with the activation status of α9ß1. Using cancer cell lines with naturally occuring high levels of this integrin, activation by α9ß1-specific ligands led to upregulation of fibronectin matrix assembly and tyrosine phosphorylation of cortactin on tyrosine 470 (Y470). Specifically, cortactin phosphorylated on Y470, but not Y421, redistributed together with α9ß1 to focal adhesions where active ß1 integrin also localises, upon integrin activation. This was commensurate with reduced migration. The localisation and phosphorylation of cortactin Y470 was regulated by Yes kinase and PTEN phosphatase. Cortactin levels influenced fibronectin matrix assembly and active ß1 integrin on the cell surface, being inversely correlated with migratory behaviour. This study underlines the complex interplay between cortactin and α9ß1 integrin that regulates cell-extracellular matrix interactions.

Phospholipase C δ1 in macrophages negatively regulates TLR4-induced proinflammatory cytokine production and Fcγ receptor-mediated phagocytosis.

Macrophages are key players in the innate immune response. Turnover of phosphoinositides (PI), particularly phosphatidylinositol 4,5 bisphosphate (PI(4,5)P2), has been implicated in macrophage functions such as toll-like receptor (TLR)-mediated cytokine production and phagocytosis. However, PI metabolizing enzymes responsible for macrophage functions are not well defined. The phospholipase C (PLC) family of enzymes is critical in PI(4,5)P2 turnover. In this study, we investigated the role of PLCδ1, a prototype PLC, in macrophages on the expression of inflammation-associated genes and phagocytosis. Lipopolysaccharides (LPS) signal through TLR4 to produce proinflammatory cytokines such as interleukin (IL)-1ß. LPS stimulation of both RAW264.7 murine macrophages and murine bone marrow-derived macrophages resulted in lower PLCδ1 mRNA and protein expression levels, compared to that in the control. Using chemical inhibitor compounds, we demonstrated that the up-regulation of p38 MAPK activity led to down-regulation of PLCδ1 mRNA expression in macrophages. PLCδ1 reduction by RNAi or gene deletion resulted in greater LPS-induced IL-1ß expression than that observed in the control siRNA-treated cells, without increasing TLR4 cell surface expression. PLCδ1 also negatively regulated LPS-induced cell spreading. Analysis of Fcγ receptor-mediated phagocytosis demonstrated an increased phagocytosis index after PLCδ1 knockdown in RAW264.7 cells. Conversely, overexpression of PLCδ1 reduced phagocytosis whereas catalytic inactive PLCδ1 had no effect. Altered levels of PLCδ1 affected the binding of opsonized latex beads with cells, rather than the phagocytic activity. Taken together, the data suggest that PLCδ1 negatively regulates LPS-induced production of IL-1ß and Fcγ receptor-mediated phagocytosis in macrophages.

Phosphorylation and mRNA splicing of collapsin response mediator protein-2 determine inhibition of rho-associated protein kinase (ROCK) II function in carcinoma cell migration and invasion.

The Rho-associated protein kinases (ROCK I and II) are central regulators of important cellular processes such as migration and invasion downstream of the GTP-Rho. Recently, we reported collapsin response mediator protein (CRMP)-2 as an endogenous ROCK II inhibitor. To reveal how the CRMP-2-ROCK II interaction is controlled, we further mapped the ROCK II interaction site of CRMP-2 and examined whether phosphorylation states of CRMP-2 affected the interaction. Here, we show that an N-terminal fragment of the long CRMP-2 splice variant (CRMP-2L) alone binds ROCK II and inhibits colon carcinoma cell migration and invasion. Furthermore, the interaction of CRMP-2 and ROCK II is partially regulated by glycogen synthase kinase (GSK)-3 phosphorylation of CRMP-2, downstream of PI3K. Inhibition of PI3K reduced interaction of CRMP-2 with ROCK II, an effect rescued by simultaneous inhibition of GSK3. Inhibition of PI3K also reduced colocalization of ROCK II and CRMP-2 at the cell periphery in human breast carcinoma cells. Mimicking GSK3 phosphorylation of CRMP-2 significantly reduced CRMP-2 binding of recombinant full-length and catalytic domain of ROCK II. These data implicate GSK3 in the regulation of ROCK II-CRMP-2 interactions. Using phosphorylation-mimetic and -resistant CRMP-2L constructs, it was revealed that phosphorylation of CRMP-2L negatively regulates its inhibitory function in ROCK-dependent haptotactic cell migration, as well as invasion of human colon carcinoma cells. Collectively, the presented data show that CRMP-2-dependent regulation of ROCK II activity is mediated through interaction of the CRMP-2L N terminus with the ROCK II catalytic domain as well as by GSK3-dependent phosphorylation of CRMP-2.

Regulation of ROCK activity in cancer.

Cancer-associated changes in cellular behavior, such as modified cell-cell contact, increased migratory potential, and generation of cellular force, all require alteration of the cytoskeleton. Two homologous mammalian serine/threonine kinases, Rho-associated protein kinases (ROCK I and II), are key regulators of the actin cytoskeleton acting downstream of the small GTPase Rho. ROCK is associated with cancer progression, and ROCK protein expression is elevated in several types of cancer. ROCKs exist in a closed, inactive conformation under quiescent conditions, which is changed to an open, active conformation by the direct binding of guanosine triphosphate (GTP)-loaded Rho. In recent years, a number of ROCK isoform-specific binding partners have been found to modulate the kinase activity through direct interactions with the catalytic domain or via altered cellular localization of the kinases. Thus, these findings demonstrate additional modes to regulate ROCK activity. This review describes the molecular mechanisms of ROCK activity regulation in cancer, with emphasis on ROCK isoform-specific regulation and interaction partners, and discusses the potential of ROCKs as therapeutic targets in cancer.

Diacylglycerol kinase ζ regulates RhoA activation via a kinase-independent scaffolding mechanism.

Rho GTPases share a common inhibitor, Rho guanine nucleotide dissociation inhibitor (RhoGDI), which regulates their expression levels, membrane localization, and activation state. The selective dissociation of individual Rho GTPases from RhoGDI ensures appropriate responses to cellular signals, but the underlying mechanisms are unclear. Diacylglycerol kinase ζ (DGKζ), which phosphorylates diacylglycerol to yield phosphatidic acid, selectively dissociates Rac1 by stimulating PAK1-mediated phosphorylation of RhoGDI on Ser-101/174. Similarly, phosphorylation of RhoGDI on Ser-34 by protein kinase Cα (PKCα) selectively releases RhoA. Here we show DGKζ is required for RhoA activation and Ser-34 phosphorylation, which were decreased in DGKζ-deficient fibroblasts and rescued by wild-type DGKζ or a catalytically inactive mutant. DGKζ bound directly to the C-terminus of RhoA and the regulatory arm of RhoGDI and was required for efficient interaction of PKCα and RhoA. DGKζ-null fibroblasts had condensed F-actin bundles and altered focal adhesion distribution, indicative of aberrant RhoA signaling. Two targets of the RhoA effector ROCK showed reduced phosphorylation in DGKζ-null cells. Collectively our findings suggest DGKζ functions as a scaffold to assemble a signaling complex that functions as a RhoA-selective, GDI dissociation factor. As a regulator of Rac1 and RhoA activity, DGKζ is a critical factor linking changes in lipid signaling to actin reorganization.

The newcomer in the integrin family: integrin α9 in biology and cancer.

Integrins are heterodimeric transmembrane receptors regulating cell-cell and cell-extracellular matrix interactions. Of the 24 integrin heterodimers identified in humans, α9ß1 integrin is one of the least studied. α9, together with α4, comprise a more recent evolutionary sub-family of integrins that is only found in vertebrates. Since α9 was thought to have similar functions as α4, due to many shared ligands, it was a rather overlooked integrin until recently, when its importance for survival after birth was highlighted upon investigation of the α9 knockout mouse. α9ß1 is expressed on a wide variety of cell types, interacts with many ligands for example fibronectin, tenascin-C and ADAM12, and has been shown to have important functions in processes such as cell adhesion and migration, lung development, lymphatic and venous valve development, and in wound healing. This has sparked an interest to investigate α9ß1-mediated signaling and its regulation. This review gives an overview of the recent progress in α9ß1-mediated biological and pathological processes, and discusses its potential as a target for cancer diagnosis and therapy.

A collapsin response mediator protein 2 isoform controls myosin II-mediated cell migration and matrix assembly by trapping ROCK II.

Collapsin response mediator protein 2 (CRMP-2) is known as a regulator of neuronal polarity and differentiation through microtubule assembly and trafficking. Here, we show that CRMP-2 is ubiquitously expressed and a splice variant (CRMP-2L), which is expressed mainly in epithelial cells among nonneuronal cells, regulates myosin II-mediated cellular functions, including cell migration. While the CRMP-2 short form (CRMP-2S) is recognized as a substrate of the Rho-GTP downstream kinase ROCK in neuronal cells, a CRMP-2 complex containing 2L not only bound the catalytic domain of ROCK II through two binding domains but also trapped and inhibited the kinase. CRMP-2L protein levels profoundly affected haptotactic migration and the actin-myosin cytoskeleton of carcinoma cells as well as nontransformed epithelial cell migration in a ROCK activity-dependent manner. Moreover, the ectopic expression of CRMP-2L but not -2S inhibited fibronectin matrix assembly in fibroblasts. Underlying these responses, CRMP-2L regulated the kinase activity of ROCK II but not ROCK I, independent of GTP-RhoA levels. This study provides a new insight into CRMP-2 as a controller of myosin II-mediated cellular functions through the inhibition of ROCK II in nonneuronal cells.

Breast and ovarian cancers: a survey and possible roles for the cell surface heparan sulfate proteoglycans.

Tumor markers are widely used in pathology not only for diagnostic purposes but also to assess the prognosis and to predict the treatment of the tumor. Because tumor marker levels may change over time, it is important to get a better understanding of the molecular changes during tumor progression. Occurrence of breast and ovarian cancer is high in older women. Common known risk factors of developing these cancers in addition to age are not having children or having children at a later age, the use of hormone replacement therapy, and mutations in certain genes. In addition, women with a history of breast cancer may also develop ovarian cancer. Here, the authors review the different tumor markers of breast and ovarian carcinoma and discuss the expression, mutations, and possible roles of cell surface heparan sulfate proteoglycans during tumorigenesis of these carcinomas. The focus is on two groups of proteoglycans, the transmembrane syndecans and the lipid-anchored glypicans. Both families of proteoglycans have been implicated in cellular responses to growth factors and morphogens, including many now associated with tumor progression.

Serine 34 phosphorylation of rho guanine dissociation inhibitor (RhoGDIalpha) links signaling from conventional protein kinase C to RhoGTPase in cell adhesion.

Conventional protein kinase C (PKC) isoforms are essential serine/threonine kinases regulating many signaling networks. At cell adhesion sites, PKCalpha can impact the actin cytoskeleton through its influence on RhoGTPases, but the intermediate steps are not well known. One important regulator of RhoGTPase function is the multifunctional guanine nucleotide dissociation inhibitor RhoGDIalpha that sequesters several related RhoGTPases in an inactive form, but it may also target them through interactions with actin-associated proteins. Here, it is demonstrated that conventional PKC phosphorylates RhoGDIalpha on serine 34, resulting in a specific decrease in affinity for RhoA but not Rac1 or Cdc42. The mechanism of RhoGDIalpha phosphorylation is distinct, requiring the kinase and phosphatidylinositol 4,5-bisphosphate, consistent with recent evidence that the inositide can activate, localize, and orient PKCalpha in membranes. Phosphospecific antibodies reveal endogenous phosphorylation in several cell types that is sensitive to adhesion events triggered, for example, by hepatocyte growth factor. Phosphorylation is also sensitive to PKC inhibition. Together with fluorescence resonance energy transfer microscopy sensing GTP-RhoA levels, the data reveal a common pathway in cell adhesion linking two essential mediators, conventional PKC and RhoA.

Heparan sulfate chain valency controls syndecan-4 function in cell adhesion.

Fibroblasts null for the transmembrane proteoglycan, syndecan-4, have an altered actin cytoskeleton, compared with matching wild-type cells. They do not organize alpha-smooth muscle actin into bundles, but will do so when full-length syndecan-4 is re-expressed. This requires the central V region of the core protein cytoplasmic domain, though not interactions with PDZ proteins. A second key requirement is multiple heparan sulfate chains. Mutant syndecan-4 with no chains, or only one chain, failed to restore the wild-type phenotype, whereas those expressing two or three were competent. However, clustering of one-chain syndecan-4 forms with antibodies overcame the block, indicating that valency of interactions with ligands is a key component of syndecan-4 function. Measurements of focal contact/adhesion size and focal adhesion kinase phosphorylation correlated with syndecan-4 status and alpha-smooth muscle actin organization, being reduced where syndecan-4 function was compromised by a lack of multiple heparan sulfate chains.

Alpha9beta1 integrin in melanoma cells can signal different adhesion states for migration and anchorage.

Cell surface integrins are the primary receptors for cell migration on extracellular matrix, and exist in several activation states regulated in part by ectodomain conformation. The alpha9 integrin subunit, which pairs only with beta1, has specific roles in the immune system and may regulate cell migration. Melanoma cells express abundant alpha9beta1 integrin, and its role in cell migration was assessed. Ligands derived from Tenascin-C and ADAM12 supported alpha9beta1 integrin-mediated cell attachment and GTP-Rac dependent migration, but not focal adhesion formation. Manganese ions induced alpha9beta1 integrin- and Rho kinase-dependent focal adhesion and stress fibre formation, suggesting that the activation status of alpha9beta1 integrin was altered. The effect of manganese ions in promoting focal adhesion formation was reproduced by beta1 integrin activating antibody. The alpha9beta1 integrin translocated to focal adhesions, where active beta1 integrin was also detected by conformation-specific antibodies. Focal adhesion assembly was commensurate with reduced cell migration. Endogenous alpha9beta1 integrin-mediated adhesion was sensitive to the PP1 chemical inhibitor and an inhibitor of endosomal vesicle recycling, but not inhibitors of protein kinase C or the small GTPase Rho. Our results demonstrated that although alpha9beta1 integrin can induce and localise to focal adhesions in a high activation state, its intermediate activity state normally supports cell adhesion consistent with migration.

Fibronectin matrix assembly requires distinct contributions from Rho kinases I and -II.

Extracellular matrix is integral to tissue architecture and regulates many aspects of cell behavior. Fibronectin matrix assembly involves the actin cytoskeleton and the small GTPase RhoA, but downstream signaling is not understood. Here, down-regulation of either rho kinase isoform (ROCK I or -II) by small interfering RNA treatment blocked fibronectin matrix assembly, although the phenotypes were distinct and despite persistence of the alternate kinase. Remnant fibronectin on ROCK-deficient fibroblasts was mostly punctate and more deoxycholate soluble compared with controls. Fibronectin matrix assembly defects in ROCK-deficient cells did not result from decreased synthesis/secretion, altered fibronectin mRNA splicing, metalloproteinase activity, or alpha5beta1 integrin dysfunction. Rescue could be effected by ROCK protein restoration or phosphomimetic myosin light chain expression. However, the effect of ROCK I deficiency on fibronectin matrix assembly was secondary to altered cell surface morphology, rich in filopodia, resulting from high GTP-Cdc42 levels. Total internal reflection microscopy revealed that a submembranous pool of myosin light chain in control cells was missing in ROCK II-deficient cells and replaced by stress fibers. Together, two rho kinases contribute to fibronectin matrix assembly in a different manner and cortical myosin II-driven contractility, but not stress fibers, may be critical in this activity.

PKCbeta-dependent activation of RhoA by syndecan-4 during focal adhesion formation.

Syndecan-4 is a ubiquitously expressed transmembrane heparan sulphate proteoglycan acting in concert with integrins in the formation of focal adhesions and stress fibres. Signalling events studied thus far suggest the formation of a ternary complex between syndecan-4, phosphatidylinositol 4,5-bisphosphate and protein kinase C alpha (PKCalpha). Syndecan-4 clustering at the cell surface has also been associated with RhoA-dependent signalling, but the relationship between PKCalpha and RhoA has not been resolved. Here we present evidence that syndecan-4, PKCalpha and RhoA are in a linear pathway necessary for the formation and maintenance of stress fibres in primary rat embryo fibroblasts. Inhibition of PKCalpha activity through the use of specific pharmacological inhibitors, a dominant-negative construct, or siRNA downregulation of protein levels, attenuated focal adhesion formation and the maintenance of stress fibres. However, these effects could be bypassed through independent activation of RhoA with lysophosphatidic acid, but not by clustering of syndecan-4 with ligand. Furthermore, inhibition of PKCalpha could block the increase in the GTP levels of RhoA induced by clustering of syndecan-4 at the cell surface. All these data point to a mechanism whereby syndecan-4 signals to RhoA in a PKCalpha-dependent manner and PKCalpha directly influences RhoA activity.