Search

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.

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 ZIC5is strongly expressed in PDAC and CCA tissues, while ZIC5expression is barely observed in most normal human adult tissues. Furthermore, ZIC5expression is related to poor prognosis of patients with PDAC. ZIC5knockdown 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, ZIC5knockdown induced cell death regardless of STAT3 activation, which is promoted by interleukin (IL) −6, a factor associated with inflammation. Furthermore, knockdown of ZIC5in 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.

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.

The dissociation of individual Rho GTPases from RhoGDI ensures appropriate responses to cellular signals. The enzyme diacylglycerol kinase ζ (DGKζ) serves as a scaffold to assemble a signaling complex that functions as a RhoA-specific RhoGDI dissociation factor. DGKζ deficiency impairs RhoA activation and stress fiber formation in fibroblasts.

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.

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 α5β1 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.

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 α5β1 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.

Background : Vitronectins (VN) are multifunctional adhesive glycoproteins that are present in plasma and the extracellular matrix of most tissues. We previously reported that the collagen-binding activity of VN is enhanced by a change in glycosylation in vitro and during liver regeneration after partial hepatectomy in vivo [Glycobiology 10 (2000) 865]. To elucidate the glycan modulation, changes of VN in cirrhosis were studied. Methods : VNs purified from patients' and normal plasma were examined for plasma concentration, reactivities against antibodies and lectins by dot blotting, carbohydrate composition, and collagen-binding activity by ELISA. Results : Plasma concentrations of VN declined in chronic liver diseases in the order of hepatitis>cirrhosis>hepatocellular carcinoma with cirrhosis. Lectin reactivities and carbohydrate analyses of VN from cirrhotic plasma (LC-VN) indicated that sialylation was elevated. LC-VN exhibited decreased binding to type I collagen. Collagen-binding studies of plasma before and after urea-treatment indicated that the active form of VN increased in cirrhotic plasma. Conclusions : The attenuated collagen-binding activity of LC-VN is attributable to a change of glycosylation. The increase of active VN in cirrhotic plasma may contribute to the matrix incorporation of VN and subsequent repair or remodeling processes.

Complementary DNA encoding mouse alpha-1,6-fuc-osyltransferase (FUT) was cloned. The deduced primary structure consisted of 575 amino acids and had 96.0%, and 93.0% identity with alpha-1,6-FUT of human and porcine origin, respectively. Quantitative analysis of alpha-1,6-FUT mRNA expression during selected developmental stages of the cerebrum showed that the expression increased during later embryonic stages and was highest in the early postnatal stages (P1 to P7), after which it declined somewhat but still remained relatively high in the mature adult. The expression profile suggests important roles of FUT in the developing central nervous system.Database. Accession No.: AB025198

We developed a method for introducing an N-glycosylation cassette into proteins at random sites by constructing cDNAs and expressing it in mammalian cells. The protocol entails four steps: (i) generation of cDNAs that contain single, randomly-located blunt end cuts; (ii)ligation of N-glycosylation cassettes into the blunt end cuts in three-frame formats; (iii) selection of the cDNA clones encoding N-glycosylated proteins; and (iv) subcloning into an expression vector for transfection and expression in mammalian cells. This method was evaluated using secreted fibroblast growth factor (FGF) as a model protein. Several secreted FGF cDNA clones, each containing an AsnLeuSer-coding sequence at a random site, were obtained. When these clones were expressed in mammalian cells, some of the secreted FGFs were found to be N-glycosylated. The method described here should also be applicable for random introduction of functional oligopeptide/polypeptide cassettes into virtually any protein of interest.

The gene for fibroblast growth factor (FGF)-6/hst-2 was originally identified by its close homology with the FGF-4/hst-1 gene. Aside from its ability to transform cultured fibroblasts, the characteristics of FGF-6 protein have only been studied using a simple preparation from E. coli. In the present study, we expressed FGF-6 cDNA in CHO cells and characterized the resultant protein. We found that CHO cells secreted several forms of the FGF-6 polypeptide, and that there were multiple N-terminal modifications. The longest form (18-kDa) contained the sequence, SerProAlaGlyAlaArg, as its N-terminus, which was consistent with the signal peptide cleavage site predicted from its primary structure. The core polypeptide was primarily modified by heterogeneous N-glycans that were sialylated to a small degree; among them, biantennary structures were found to predominate. Moreover, possible O-glycosylation was also detected. N-glycosylated FGF-6 potently induced DNA synthesis and proliferation of human vascular endothelial cells, whereas in the absence of N-glycosylation, FGF-6 mitogenicity was substantially diminished. The results clearly indicate that FGF-6 expressed by mammalian cells is a glycosylated mitogen for vascular endothelial cells and further suggests that N-glycosylation plays a key role in determining the mitogenicity of FGF-6.

We detected in the brain and then cloned two novel, short forms of human and mouse fibroblast growth factor (FGF)-5 mRNA, which were designated human FGF-5S (hFGF-5S) and mouse FGF-5S (mFGF-5S), respectively. Genomic analysis indicated that mFGF-5S and authentic mFGF-5 mRNAs were transcribed from a single gene; hFGF-5S and mFGF-5S mRNAs were generated by excluding the second exon of the respective FGF-5 genes, and the alternatively spliced mRNAs encoded for 123- and 121-amino acid proteins, respectively. Indeed, a neuron-like cell line expressing mFGF-5S mRNA secreted a protein of the expected size and with FGF-5 antigenicity. In PC12 cells, expression of hFGF-5 or exposure to hFGF-5 protein induced differentiation. Neither expression of hFGF-5S, alone, nor co-expression of hFGF-5S with hFGF-5 induced significant differentiation. At high concentrations, hFGF-5S protein partially antagonized FGF-5 activity, whereas by itself, hFGF-5S exerted very weak neurotrophic activity. hFGF-5S protein binds to FGF receptor (FGFR)-1 on PC12 transfectants and partially inhibits hFGF-5-induced tyrosine phosphorylation of FGFR-1 and an FGFR substrate, but it also induces phosphorylation by itself. These results suggest that FGF-5S is a naturally expressed partial agonist/antagonist of FGF-5 neurotrophic activity in the brain and that its effects are exerted in part at the level of the receptor.