Molecular mechanisms and effects of a rasgap-derived peptide on metastatic progression

Cancer is the second leading cause of mortality worldwide. Cancer progression leads to metastasis formation, which accounts for more than ninety percent of cancer-related death. Metastases are more difficult to be surgically removed because of their invasive behavior and shape. In addition, during their transformation journey, they become more and more resistant to anticancer drugs. Significant improvements have been achieved in therapy against cancer in recent years but targeting the metastatic cascade remains the Achilles heel of the cure against cancer. A First step in the metastatic process is the escape of cancer cells from the primary tumor site. This involves an increase in cell motility and the concomitant ability to clear a path through the extracellular matrix. From a therapeutic point of view, inhibition of cell migration is a logical approach to develop anti-metastatic drugs. Our lab previously developed a cell permeable peptide derived from a caspase-3-generaied fragment of the RasGAP protein called TAT-RasGAP317-326. This peptide efficiently and specifically sensitizes cancer cells to chemotherapy- and radiotherapy-induced ceil death, which allows decreasing the anticancer drug doses and eventually their associated side- effects. In the present study we discovered that TAT-RasGAP317.326 also increases cell adhesion which was associated with inhibition of cell migration and invasion into the extracellular matrix. The ability of TAT-RasGAP317.326 to increase ceil adhesion involves the dramatic depolymerization of actin cytoskekton together with redistribution of focal adhesions. We found that the inhibitory effects on migration were mediated by a RhoGAP tumor and metastasis suppressor cailed DLC1 (Deleted in Liver Cancer 1). Moreover. DEC 1 was found to be a direct RasGAP-interacting protein and this interaction requires the RasGAP tryptophan 317 residue, the very first RasGAP residue of TAT-RasGAP317.326. We then evaluated the roie of RasGAP fragments in the in vivo metastatic cascade. We found that breast cancer cells overexpressing the parental RasGAP fragment, to which the TAT-RasGAP317.326 peptide belongs, have a markedly decreased ability to form lung metastases. Unfortunately, we were not able to recapitulate these an ti-metastatic effects when TAT-RasGAP317.326 was injected. However, we later understood that this was due to the fact that TAT-RasGAP317.326 was not properly delivered to the primary tumors. Further work, aimed at better understanding of how TAT-RasGAP317.326 functions, revealed that the ten amino acid TAT-RasGAP317.326 peptide could, be narrowed down to a three amino acid TAT-RasGAP317.329 peptide while keeping its sensitizer activity. In parallel, investigations on the RasGAP-DLCl binding indicated that the arginine linger of the DLC1 GAP domain is required for this interaction, which suggests that TAT-RasGAP317.326 modulates the GAP activity of DLC1. Additional work should be performed to fully elucidate its mechanism of action and render TAT-RasGAP317.326 usable as a tool to fight cancer on two fronts, by improving chemotherapy and preventing metastatic progression. - Le cancer est la deuxième cause de mortalité dans le monde. La formation de métastases est la dernière étape de la progression cancéreuse et représente plus du nonante pour cent des morts induites par le cancer. De par leur morphologie et comportement invasifs, ii est difficile d'avoir recours à la chirurgie pour exciser des métastases. De plus, les cellules cancéreuses en progression deviennent souvent de plus en plus résistantes aux drogues anticancéreuses. Ces dernières années, des avancements significatifs ont contribué à l'amélioration de la lutte contre le cancer. Néanmoins, pouvoir cibler spécifiquement la cascade métastatique demeure cependant le talon d'Achille des thérapies anticancéreuses. Une première étape dans ie processus métastatique est l'évasion des cellules cancéreuses du site de la tumeur primaire. Ceci requiert une augmentation de la motiliié cellulaire couplée à la capacité de se frayer un chemin au sein de la matrice extracelluiaire. D'un point de vue thérapeutique, inhiber la migration cellulaire est une approche attrayante. Notre laboratoire a développé un peptide, nommé TAT-RasGAP317.326 dérivé d'un fragment qui est lui-même le résultat du clivage de la protéine RasGAP par la caspase-3. Ce peptide est capable de pénétrer les cellules cancéreuses et de les sensibiliser spécifiquement à la mort induite par la radiothérapie et la chimiothérapie. La finalité des effets de ce peptide est de pouvoir diminuer les doses des traitements anti-cancéreux et donc des effets secondaires qu'ils engendrent. Dans cette étude, nous avons découvert que TAT-RasGAP317.326 augmente l'adhésion des cellules et inhibe la migration cellulaire ainsi que l'invasion des cellules à travers une matrice extracellulaire. La capacité de TAT-RasGAP317.326 à induire l'adhésion repose sur ia dépolymérisation du cytosquelette d'actine associée à une redistribution des points d'ancrage cellulaire. Nous avons découvert que l'inhibition de ia migration par TAT-RasGAP317.326 nécessitait la présence d'un suppresseur de tumeur et de métastases appelé DLC1 (Deleted in Liver Cancer l), qui par ailleurs s'avère aussi être une protéine RhoGAP. De plus, nous avons aussi trouvé que DLC1 était un partenaire d'interaction de RasGAP et que cette interaction s'effectuait via l'acide aminé tryptophane 317 de RasGAP. qui s'avère être le premier acide aminé du peptide TAT-RasGAP317.326. Nous avons ensuite évalué le rôle joué par certains fragments de RasGAP dans le processus de métastatisation. Dans ce contexte, des cellules de cancer du sein qui sur-expriment un fragment de RasGAP contenant la séquence TAT-RasGAP317.326 ont vu leur potentiel métastatique diminuer drastiquerment. Malheureusement, aucun effet anti-métastatique n'a été obtenu après injection de TAT-RasGAP317.326 dans les souris. Cependant, nous avons réalisé rétrospectivement que TAT-RasGAP317.326 n'était pas correctement délivré à la tumeur primaire, ce qui nous empêche de tirer des conclusions sur le rôle anti-métastatique de ce peptide. La suite de cette étude visant à mieux comprendre comment TAT-RasGAP317.326 agit, a mené à la découverte que les dix acides aminés de TAT-RasGAP317.326 pouvaient être réduits à trois acides aminés, TAT-RasGAP317.329, tout en gardant l'effet sensibilisateur à la chimiothérapie. En visant à élucider le mode d'interaction entre RasGAP et DLC1, nous avons découvert qu'un acide aminé nécessaire à l'activité GAP de DLC1 était requis pour lier RasGAP, ce qui laisse présager que TAT-RasGAp317.32c, module i'activité GAP de DLC1. Des travaux supplémentaires doivent encore être effectués pour complètement élucider les mécanismes d'action de TAT-RasGAP317.326 et afin de pouvoir l'utiliser comme un outil pour combattre le cancer sur deux fronts, en améliorant les chimiothérapies et en inhibant la formation de métastases.

Differential phosphorylation of some proteins of the neuronal cytoskeleton during brain development.

The cytoskeleton is important for neuronal morphogenesis. During the postnatal development of cat brain, the molecular composition of the neuronal cytoskeleton changes with maturation. Several of its proteins change in their rate of expression, in their degree of phosphorylation, in their subcellular distribution, or in their biochemical properties. It is proposed that phosphorylation is an essential mechanism to regulate the plasticity of the early, juvenile-type cytoskeleton. Among such proteins are several microtubule-associated proteins (MAPs), such as MAP5a, MAP2c or the juvenile tau proteins. Phosphorylation may also act on neurofilaments, postulated to be involved in the adult-type stabilization of axons. These observations imply that phosphorylation may affect cytoskeleton function in axons and dendrites at various developmental stages. Yet, the mechanisms of phosphorylation and its regulation cascades are largely unknown. In view of the topic of this issue on CD15, the potential role of matrix molecules being involved in the modulation of phosphorylation activity and of cytoskeletal properties is addressed.

Unavailability of CD147 leads to selective erythrocyte trapping in the spleen.

Adhesive interactions with stromal cells and the extracellular matrix are essential for the differentiation and migration of hematopoietic progenitors. In the erythrocytic lineage, a number of adhesion molecules are expressed in the developing erythrocytes and are thought to play a role in the homing and maturation of erythrocytic progenitors. However, many of these molecules are lost during the final developmental stages leading to mature erythrocytes. One of the adhesion molecules that remains expressed in mature, circulating erythrocytes is CD147. This study shows that blockade of this molecule on the cell surface by treatment with F(ab')(2) fragments of anti-CD147 monoclonal antibody disrupts the circulation of erythrocytes, leading to their selective trapping in the spleen. Consequently, mice develop an anemia, and de novo, erythropoietin-mediated erythropoiesis in the spleen. In contrast, these changes were not seen in mice similarly treated with another antierythrocyte monoclonal antibody with a different specificity. These results suggest that the CD147 expressed on erythrocytes likely plays a critical role in the recirculation of mature erythrocytes from the spleen into the general circulation. (Blood. 2001;97:3984-3988)

In-depth analysis of hyaline fibromatosis syndrome frameshift mutations at the same site reveal the necessity of personalized therapy.

Hyaline fibromatosis syndrome is an autosomal recessive disease caused by mutations in ANTXR2, a gene involved in extracellular matrix homeostasis. Sixty percent of patients carry frameshift mutations at a mutational hotspot in exon 13. We show in patient cells that these mutations lead to low ANTXR2 mRNA and undetectable protein levels. Ectopic expression of the proteins encoded by the mutated genes reveals that a two base insertion leads to the synthesis of a protein that is rapidly targeted to the ER-associated degradation pathway due to the modified structure of the cytosolic tail, which instead of being hydrophilic and highly disordered as in wild type ANTXR2, is folded and exposes hydrophobic patches. In contrast, one base insertion leads to a truncated protein that properly localizes to the plasma membrane and retains partial function. We next show that targeting the nonsense mediated mRNA decay pathway in patient cells leads to a rescue of ANTXR2 protein in patients carrying one base insertion but not in those carrying two base insertions. This study highlights the importance of in-depth analysis of the molecular consequences of specific patient mutations, which even when they occur at the same site can have drastically different consequences.

Tenascin-W is a specific marker of glioma-associated blood vessels and stimulates angiogenesis in vitro.

The microenvironment hosting a tumor actively participates in regulating tumor cell proliferation, migration, and invasion. Among the extracellular matrix proteins enriched in the stroma of carcinomas are the tenascin family members tenascin-C and tenascin-W. Whereas tenascin-C overexpression in gliomas is known to correlate with poor prognosis, the status of tenascin-W in brain tumors has not been investigated so far. In the present study, we analyzed protein levels of tenascin-W in 38 human gliomas and found expression of tenascin-W in 80% of the tumor samples, whereas no tenascin-W could be detected in control, nontumoral brain tissues. Double immunohistochemical staining of tenascin-W and von Willebrand factor revealed that tenascin-W is localized around blood vessels, exclusively in tumor samples. In vitro, the presence of tenascin-W increased the proportion of elongated human umbilical vein endothelial cells (HUVECs) and augmented the mean speed of cell migration. Furthermore, tenascin-W triggered sprouting of HUVEC spheroids to a similar extent as the proangiogenic factor tenascin-C. In conclusion, our study identifies tenascin-W as a candidate biomarker for brain tumor angiogenesis that could be used as a molecular target for therapy irrespective of the glioma subtype.-Martina, E., Degen, M., Rüegg, C., Merlo, A., Lino, M. M., Chiquet-Ehrismann, R., Brellier, F. Tenascin-W is a specific marker of glioma-associated blood vessels and stimulates angiogenesis in vitro.

Peroxisome proliferator-activated receptor-beta signaling contributes to enhanced proliferation of hepatic stellate cells.

BACKGROUND & AIMS: The peroxisome proliferator-activated nuclear receptors (PPAR-alpha, PPAR-beta, and PPAR-gamma), which modulate the expression of genes involved in energy homeostasis, cell cycle, and immune function, may play a role in hepatic stellate cell activation. Previous studies focused on the decreased expression of PPAR-gamma in hepatic stellate cell activation but did not investigate the expression and role of the PPAR-alpha and -beta isotypes. The aim of this study was to evaluate the expression of the different PPARs during hepatic stellate cell activation in vitro and in situ and to analyze possible factors that might contribute to their expression. In a second part of the study, the effect of a PPAR-beta agonist on acute liver injury was evaluated. METHODS: The effects of PPAR isotype-specific ligands on hepatic stellate cell transition were evaluated by bromodeoxyuridine incorporation, gel shifts, immunoprecipitation, and use of antisense PPAR-beta RNA-expressing adenoviruses. Tumor necrosis factor alpha-induced PPAR-beta phosphorylation and expression was evaluated by metabolic labeling and by using specific P38 inhibitors. RESULTS: Hepatic stellate cells constitutively express high levels of PPAR-beta, which become further induced during culture activation and in vivo fibrogenesis. No significant expression of PPAR-alpha or -gamma was found. Stimulation of the P38 mitogen-activated protein kinase pathway modulated the expression of PPAR-beta. Transcriptional activation of PPAR-beta by L165041 enhanced hepatic stellate cell proliferation. Treatment of rats with a single bolus of CCl(4) in combination with L165041 further enhanced the expression of fibrotic markers. CONCLUSIONS: PPAR-beta is an important signal-transducing factor contributing to hepatic stellate cell proliferation during acute and chronic liver inflammation.

Ultrastructural and molecular analysis of Bowman's layer corneal dystrophies: an epithelial origin?

PURPOSE: Two mutations (R555Q and R124L) in the BIGH3 gene have been described in anterior or Bowman's layer dystrophies (CDB). The clinical, molecular, and ultrastructural findings of five families with CDB was reviewed to determine whether there is a consistent genotype:phenotype correlation. METHODS: Keratoplasty tissue from each patient was examined by light and electron microscopy (LM and EM). DNA was obtained, and exons 4 and 12 of BIGH3 were analyzed by polymerase chain reaction and single-stranded conformation polymorphism/heteroduplex analysis. Abnormally migrating products were analyzed by direct sequencing. RESULTS: In two families with type I CDB (CDBI), the R124L mutation was defined. There were light and ultrastructural features of superficial granular dystrophy and atypical banding of the "rod-shaped bodies" ultrastructurally. Patients from three families with "honeycomb" dystrophy were found to carry the R555Q mutation and had characteristic features of Bowman's dystrophy type II (CDBII). CONCLUSIONS: There is a strong genotype:phenotype correlation among CBDI (R124L) and CDBII (R555Q). LM and EM findings suggest that epithelial abnormalities may underlie the pathology of both conditions. The findings clarify the confusion over classification of the Bowman's layer dystrophies.

Role of innate immunity in cardiac inflammation after myocardial infarction.

Over the past two decades, inflammation has emerged as a key pathophysiological process during myocardial infarction. It develops consecutively to the activation of innate immune defense mechanisms, in response to the release of endogenous molecules by necrotic cells and the extracellular matrix. These danger signals are sensed by cellular receptors normally involved in antimicrobial defenses, including toll-like receptors and a subset of NOD-like receptors, which promote intracellular signaling dependent on nuclear factor kappaB and on the formation of the inflammasome. These mechanisms stimulate the expression of multiple inflammatory mediators and growth factors, sequentially inducing the recruitment of inflammatory cells, the clearance of injured tissue, angiogenesis, and the proliferation of fibroblasts, eventually resulting in scar formation and infarct healing. Dysregulation of these responses may result in continued cardiomyocyte loss, fibrosis beyond the limits of the infarcted area, reactive hypertrophy and chamber dilatation, a process termed adverse cardiac remodeling, leading to functional compromise and heart failure. This review presents the current state of knowledge on the process of immune activation within the infarcted myocardium and its consequences.

Cell entry of Lassa virus induces tyrosine phosphorylation of dystroglycan.

The extracellular matrix (ECM) receptor dystroglycan (DG) serves as a cellular receptor for the highly pathogenic arenavirus Lassa virus (LASV) that causes a haemorrhagic fever with high mortality in human. In the host cell, DG provides a molecular link between the ECM and the actin cytoskeleton via the adapter proteins utrophin or dystrophin. Here we investigated post-translational modifications of DG in the context of LASV cell entry. Using the tyrosine kinase inhibitor genistein, we found that tyrosine kinases are required for efficient internalization of virus particles, but not virus-receptor binding. Engagement of cellular DG by LASV envelope glycoprotein (LASV GP) in human epithelial cells induced tyrosine phosphorylation of the cytoplasmic domain of DG. LASV GP binding to DG further resulted in dissociation of the adapter protein utrophin from virus-bound DG. This virus-induced dissociation of utrophin was affected by genistein treatment, suggesting a role of receptor tyrosine phosphorylation in the process.

Complex genetics in idiopathic hypogonadotropic hypogonadism.

Idiopathic hypogonadotropic hypogonadism (IHH) is an important human disease model. Investigations of the genetics of IHH have facilitated insights into critical pathways regulating sexual maturation and fertility. IHH has been traditionally considered a monogenic disorder. This model holds that a single gene defect is responsible for the disease in each patient. In the case of IHH, 30% of cases are explained by mutations in one of eleven genes. In recent years, several lines of evidence have challenged the monogenic paradigm in IHH. First, disease-associated mutations display striking incomplete penetrance and variable expressivity within and across IHH families. Second, each locus is responsible for only a small percentage of cases. Third, more than one disease-associated mutation seems to be segregating in some families with IHH, and their combined or separate presence in individuals accounts for the variability in disease severity. Finally, IHH is not strictly a congenital and life-long disorder; occasionally it manifests itself during adulthood (adult-onset IHH); in other cases, the disease is not permanent, as evidenced by normal activity of the hypothalamic-pituitary-gonadal axis after discontinuation of treatment in adulthood (IHH reversal). Together, these observations suggest that IHH is not strictly a monogenic mendelian disease, as previously thought. Rather, it is emerging as a digenic, and potentially oligogenic disease, in which hormonal and/or environmental factors may critically influence genetic predisposition and clinical course. Future investigations of IHH should characterize the extent of the involvement of multiple genes in disease pathogenesis, and elucidate the contributions of epigenetic factors.

A functional and structural study of the major metalloprotease secreted by the pathogenic fungus Aspergillus fumigatus.

Fungalysins are secreted fungal peptidases with the ability to degrade the extracellular matrix proteins elastin and collagen and are thought to act as virulence factors in diseases caused by fungi. Fungalysins constitute a unique family among zinc-dependent peptidases that bears low sequence similarity to known bacterial peptidases of the thermolysin family. The crystal structure of the archetype of the fungalysin family, Aspergillus fumigatus metalloprotease (AfuMep), has been obtained for the first time. The 1.8 Å resolution structure of AfuMep corresponds to that of an autoproteolyzed proenzyme with separate polypeptide chains corresponding to the N-terminal prodomain in a binary complex with the C-terminal zinc-bound catalytic domain. The prodomain consists of a tandem of cystatin-like folds whose C-terminal end is buried into the active-site cleft of the catalytic domain. The catalytic domain harbouring the key catalytic zinc ion and its ligands, two histidines and one glutamic acid, undergoes a conspicuous rearrangement of its N-terminal end during maturation. One key positively charged amino-acid residue and the C-terminal disulfide bridge appear to contribute to its structural-functional properties. Thus, structural, biophysical and biochemical analysis were combined to provide a deeper comprehension of the underlying properties of A. fumigatus fungalysin, serving as a framework for the as yet poorly known metallopeptidases from pathogenic fungi.

Pathways for the synthesis of polyesters in plants; cutin, suberin, and polyhydroxyalkanoates

Plants naturally produce the lipid-derived polyester cutin, which is found in the plant cuticle that is deposited at the outermost extracellular matrix of the epidermis covering nearly all aboveground tissues. Being at the interface between the cell and the external environment, cutin and the cuticle play important roles in the protection of plants from several stresses. A number of enzymes involved in the synthesis of cutin monomers have recently been identified, including several P450s and one acyl-CoA synthetase, thus representing the first steps toward the understanding of polyester formation and, potentially, polyester engineering to improve the tolerance of plants to stresses, such as drought, and for industrial applications. However, numerous processes underlying cutin synthesis, such as a controlled polymerization, still remain elusive. Suberin is a second polyester found in the extracellular matrix, most often synthesized in root tissues and during secondary growth. Similar to cutin, the function of suberin is to seal off the respective tissue to inhibit water loss and contribute to resistance to pathogen attack. Being the main constituent of cork, suberin is a plant polyester that has already been industrially exploited. Genetic engineering may be worth exploring in order to change the polyester properties for either different applications or to increase cork production in other species. Polyhydroxyalkanoates (PHAs) are attractive polyesters of 3-hydroxyacids because of their properties as bioplastics and elastomers. Although PHAs are naturally found in a wide variety of bacteria, biotechnology has aimed at producing these polymers in plants as a source of cheap and renewable biodegradable plastics. Synthesis of PHA containing various monomers has been demonstrated in the cytosol, plastids, and peroxisomes of plants. Several biochemical pathways have been modified in order to achieve this, including the isoprenoid pathway, the fatty acid biosynthetic pathway, and the fatty acid β-oxidation pathway. PHA synthesis has been demonstrated in a number of plants, including monocots and dicots, and up to 40% PHA per gram dry weight has been demonstrated in Arabidopsis thaliana. Despite some successes, production of PHA in crop plants remains a challenging project. PHA synthesis at high level in vegetative tissues, such as leaves, is associated with chlorosis and reduced growth. The challenge for the future is to succeed in synthesis of PHA copolymers with a narrow range of monomer compositions, at levels that do not compromise plant productivity. This goal will undoubtedly require a deeper understanding of plant biochemical pathways and how carbon fluxes through these pathways can be manipulated, areas where plant "omics" can bring very valuable contributions.

Living-engineered valves for transcatheter venous valve repair.

Background: Chronic venous insufficiency (CVI) represents a major global health problem with increasing prevalence and morbidity. CVI is due to an incompetence of the venous valves, which causes venous reflux and distal venous hypertension. Several studies have focused on the replacement of diseased venous valves using xeno- and allogenic transplants, so far with moderate success due to immunologic and thromboembolic complications. Autologous cell-derived tissue-engineered venous valves (TEVVs) based on fully biodegradable scaffolds could overcome these limitations by providing non-immunogenic, non-thrombogenic constructs with remodeling and growth potential. Methods: Tri- and bicuspid venous valves (n=27) based on polyglycolic acid-poly-4-hydroxybutyrate composite scaffolds, integrated into self-expandable nitinol stents, were engineered from autologous ovine bone-marrow-derived mesenchymal stem cells (BM-MSCs) and endothelialized. After in vitro conditioning in a (flow) pulse duplicator system, the TEVVs were crimped (n=18) and experimentally delivered (n=7). The effects of crimping on the tissue-engineered constructs were investigated using histology, immunohistochemistry, scanning electron microscopy, grating interferometry (GI), and planar fluorescence reflectance imaging. Results: The generated TEVVs showed layered tissue formation with increasing collagen and glycosaminoglycan levels dependent on the duration of in vitro conditioning. After crimping no effects were found on the MSC level in scanning electron microscopy analysis, GI, histology, and extracellular matrix analysis. However, substantial endothelial cell loss was detected after the crimping procedure, which could be reduced by increasing the static conditioning phase. Conclusions: Autologous living small-diameter TEVVs can be successfully fabricated from ovine BM-MSCs using a (flow) pulse duplicator conditioning approach. These constructs hold the potential to overcome the limitations of currently used non-autologous replacement materials and may open new therapeutic concepts for the treatment of CVI in the future.

Thy-1-mediated cell-cell contact induces astrocyte migration through the engagement of αVβ3 integrin and syndecan-4.

Cell adhesion to the extracellular matrix proteins occurs through interactions with integrins that bind to Arg-Gly-Asp (RGD) tripeptides, and syndecan-4, which recognizes the heparin-binding domain of other proteins. Both receptors trigger signaling pathways, including those that activate RhoGTPases such as RhoA and Rac1. This sequence of events modulates cell adhesion to the ECM and cell migration. Using a neuron-astrocyte model, we have reported that the neuronal protein Thy-1 engages αVβ3 integrin and syndecan-4 to induce RhoA activation and strong astrocyte adhesion to their underlying substrate. Thus, because cell-cell interactions and strong cell attachment to the matrix are considered antagonistic to cell migration, we hypothesized that Thy-1 stimulation of astrocytes should preclude cell migration. Here, we studied the effect of Thy-1 expressing neurons on astrocyte polarization and migration using a wound-healing assay and immunofluorescence analysis. Signaling molecules involved were studied by affinity precipitation, western blotting and the usage of specific antibodies. Intriguingly, Thy-1 interaction with its two receptors was found to increase astrocyte polarization and migration. The latter events required interactions of these receptors with both the RGD-like sequence and the heparin-binding domain of Thy-1. Additionally, prolonged Thy-1-receptor interactions inhibited RhoA activation while activating FAK, PI3K and Rac1. Therefore, sustained engagement of integrin and syndecan-4 with the neuronal surface protein Thy-1 induces astrocyte migration. Interestingly we identify here, a cell-cell interaction that despite initially inducing strong cell attachment, favors cell migration upon persistent stimulation by engaging the same signaling receptors and molecules as those utilized by the extracellular matrix proteins to stimulate cell movement.

Lethal skeletal dysplasia in mice and humans lacking the golgin GMAP-210.

BACKGROUND: Establishing the genetic basis of phenotypes such as skeletal dysplasia in model organisms can provide insights into biologic processes and their role in human disease. METHODS: We screened mutagenized mice and observed a neonatal lethal skeletal dysplasia with an autosomal recessive pattern of inheritance. Through genetic mapping and positional cloning, we identified the causative mutation. RESULTS: Affected mice had a nonsense mutation in the thyroid hormone receptor interactor 11 gene (Trip11), which encodes the Golgi microtubule-associated protein 210 (GMAP-210); the affected mice lacked this protein. Golgi architecture was disturbed in multiple tissues, including cartilage. Skeletal development was severely impaired, with chondrocytes showing swelling and stress in the endoplasmic reticulum, abnormal cellular differentiation, and increased cell death. Golgi-mediated glycosylation events were altered in fibroblasts and chondrocytes lacking GMAP-210, and these chondrocytes had intracellular accumulation of perlecan, an extracellular matrix protein, but not of type II collagen or aggrecan, two other extracellular matrix proteins. The similarities between the skeletal and cellular phenotypes in these mice and those in patients with achondrogenesis type 1A, a neonatal lethal form of skeletal dysplasia in humans, suggested that achondrogenesis type 1A may be caused by GMAP-210 deficiency. Sequence analysis revealed loss-of-function mutations in the 10 unrelated patients with achondrogenesis type 1A whom we studied. CONCLUSIONS: GMAP-210 is required for the efficient glycosylation and cellular transport of multiple proteins. The identification of a mutation affecting GMAP-210 in mice, and then in humans, as the cause of a lethal skeletal dysplasia underscores the value of screening for abnormal phenotypes in model organisms and identifying the causative mutations.