Muscle β1D Integrin Reinforces the Cytoskeleton–Matrix Link: Modulation of Integrin Adhesive Function by Alternative Splicing

Expression of muscle-specific β1D integrin with an alternatively spliced cytoplasmic domain in CHO and GD25, β1 integrin-minus cells leads to their phenotypic conversion. β1D-transfected nonmuscle cells display rounded morphology, lack of pseudopodial activity, retarded spreading, reduced migration, and significantly enhanced contractility compared with their β1A-expressing counterparts. The transfected β1D is targeted to focal adhesions and efficiently displaces the endogenous β1A and αvβ3 integrins from the sites of cell–matrix contact. This displacement is observed on several types of extracellular matrix substrata and leads to elevated stability of focal adhesions in β1D transfectants. Whereas a significant part of cellular β1A integrin is extractable in digitonin, the majority of the transfected β1D is digitonin-insoluble and is strongly associated with the detergent-insoluble cytoskeleton. Increased interaction of β1D integrin with the actin cytoskeleton is consistent with and might be mediated by its enhanced binding to talin. In contrast, β1A interacts more strongly with α-actinin, than β1D. Inside-out driven activation of the β1D ectodomain increases ligand binding and fibronectin matrix assembly by β1D transfectants. Phenotypic effects of β1D integrin expression in nonmuscle cells are due to its enhanced interactions with both cytoskeletal and extracellular ligands. They parallel the transitions that muscle cells undergo during differentiation. Modulation of β1 integrin adhesive function by alternative splicing serves as a physiological mechanism reinforcing the cytoskeleton– matrix link in muscle cells. This reflects the major role for β1D integrin in muscle, where extremely stable association is required for contraction.     

Sulphated glycosaminoglycans expression in the basement membranes of colorectal adenocarcinomas. Preliminary study: correlation with histological grading

Summary The expression of sulphated glycosaminoglycans was studied at the ultrastructural level by the high iron diamine technique in the basement membranes of 26 colorectal adenocarcinomas (10 well-differentiated, 7 moderately-differentiated, 9 poorlydifferentiated). Sulphated glycosaminoglycan expression was highly variable. It was scored as regular (5 cases), slightly irregular (6 cases), highly irregular (15 cases). In general, poor histological differentiation could be correlated with absent or highly irregular expression. However, in a limited number of cases, severe alterations of basement membranes were also present in well-differentiated (2 cases) and moderately-differentiated (4 cases) tumours. Such a variability shows up a heterogeneity which is not revealed by histological grading.     

β1-Integrin Cytoplasmic Subdomains Involved in Dominant Negative Function

The β1-integrin cytoplasmic domain consists of a membrane proximal subdomain common to the four known isoforms (“common” region) and a distal subdomain specific for each isoform (“variable” region). To investigate in detail the role of these subdomains in integrin-dependent cellular functions, we used β1A and β1B isoforms as well as four mutants lacking the entire cytoplasmic domain (β1TR), the variable region (β1COM), or the common region (β1ΔCOM-B and β1ΔCOM-A). By expressing these constructs in Chinese hamster ovary and β1 integrin-deficient GD25 cells (Wennerberg et al., J Cell Biol 132, 227–238, 1996), we show that β1B, β1COM, β1ΔCOM-B, and β1ΔCOM-A molecules are unable to support efficient cell adhesion to matrix proteins. On exposure to Mn⁺⁺ ions, however, β1B, but none of the mutants, can mediate cell adhesion, indicating specific functional properties of this isoform. Analysis of adhesive functions of transfected cells shows that β1B interferes in a dominant negative manner with β1A and β3/β5 integrins in cell spreading, focal adhesion formation, focal adhesion kinase tyrosine phosphorylation, and fibronectin matrix assembly. None of the β1 mutants tested shows this property, indicating that the dominant negative effect depends on the specific combination of common and B subdomains, rather than from the absence of the A subdomain in the β1B isoform.     

The Muscle-Specific Laminin Receptor α7β1 Integrin Negatively Regulates α5β1 Fibronectin Receptor Function

α7β1 is the major integrin complex expressed in differentiated muscle cells where it functions as a laminin receptor. In this work we have expressed the α7 integrin subunit in CHO cells to investigate the functional properties of this receptor. After transfection with α7 CHO cells acquired the ability to adhere and spread on laminin 1 consistent with the laminin receptor activity of the α7β1. α7 transfectants, however, showed a 70% reduction in the ability to adhere to fibronectin and were unable to assemble a fibronectin matrix. The degree of reduction was inversely related to the level of α7 expression. To define the mechanisms underlying this adhesive defect we analyzed surface expression and functional properties of the α5β1 fibronectin receptor. Although cell surface expression of α5β1 was reduced by a factor of 20–25% in α7 transfectants compared to control untransfected cells, this slight reduction was not sufficient to explain the dramatic reduction in cell adhesion (70%) and matrix assembly (close to 100%). Binding studies showed that the affinity of¹²⁵I-fibronectin for its surface receptor was decreased by 50% in α7 transfectants, indicating that the α5β1 integrin is partially inactivated in these cells. Inactivation can be reversed by Mn²⁺, a cation known to increase integrin affinity for their ligands. In fact, incubation of cells with Mn²⁺restored fibronectin binding affinity, adhesion to fibronectin, and assembly of fibronectin matrix in α7 transfectants. These data indicate that α7 expression leads to the functional down regulation of α5β1 integrin by decreasing ligand binding affinity and surface expression. In conclusion, the data reported establish the existence of anegative cooperativitybetween α7 and α5 integrins that may be important in determining functional regulation of integrins during myogenic differentiation.     

Histidines Are Responsible for Zinc Potentiation of the Current in KDC1 Carrot Channels

Unlike all plant inward-rectifying potassium channels, the carrot channel KDC1 has two histidine pairs (H161,H162) in the S3–S4 and (H224,H225) in the S5–S6 linkers. When coinjected with KAT1 in Xenopus oocytes, KDC1 participates in the formation of heteromultimeric KDC1:KAT1 channels and the ionic current is potentiated by extracellular Zn²⁺. To investigate the potential interactions between KDC1 and zinc, a KDC1-KAT1 dimer was constructed. The dimeric and heteromeric channels displayed similar characteristics and the same sensitivity to zinc and other metals; this result suggests that zinc binding is mediated by residues in a single channel subunit. The KDC1:KAT1 currents were also potentiated by external Pb²⁺ and Cd²⁺ and inhibited by Ni²⁺. To investigate further the role of KDC1-histidines, these amino acids were mutated into alanines. The single mutations H225A, H161A, and H162A did not affect the response of the heteromeric channels to zinc. Conversely, the single mutant H224A and the double mutants (H224A,H225A) and (H161A,H162A) abolished zinc potentiation, but not that induced by Pb²⁺ or Cd²⁺. These results suggest that Zn²⁺ potentiation cannot be ascribed to simple electrostatic interactions between zinc and channel residues and that histidine 224 is crucial for zinc but not for lead potentiation of the current.     

p63 Gene Mutations in EEC Syndrome, Limb-Mammary Syndrome, and Isolated Split Hand–Split Foot Malformation Suggest a Genotype-Phenotype Correlation

p63 mutations have been associated with EEC syndrome (ectrodactyly, ectodermal dysplasia, and cleft lip/palate), as well as with nonsyndromic split hand-split foot malformation (SHFM). We performed p63 mutation analysis in a sample of 43 individuals and families affected with EEC syndrome, in 35 individuals affected with SHFM, and in three families with the EEC-like condition limb-mammary syndrome (LMS), which is characterized by ectrodactyly, cleft palate, and mammary-gland abnormalities. The results differed for these three conditions. p63 gene mutations were detected in almost all (40/43) individuals affected with EEC syndrome. Apart from a frameshift mutation in exon 13, all other EEC mutations were missense, predominantly involving codons 204, 227, 279, 280, and 304. In contrast, p63 mutations were detected in only a small proportion (4/35) of patients with isolated SHFM. p63 mutations in SHFM included three novel mutations: a missense mutation (K193E), a nonsense mutation (Q634X), and a mutation in the 3' splice site for exon 5. The fourth SHFM mutation (R280H) in this series was also found in a patient with classical EEC syndrome, suggesting partial overlap between the EEC and SHFM mutational spectra. The original family with LMS (van Bokhoven et al. 1999) had no detectable p63 mutation, although it clearly localizes to the p63 locus in 3q27. In two other small kindreds affected with LMS, frameshift mutations were detected in exons 13 and 14, respectively. The combined data show that p63 is the major gene for EEC syndrome, and that it makes a modest contribution to SHFM. There appears to be a genotype-phenotype correlation, in that there is a specific pattern of missense mutations in EEC syndrome that are not generally found in SHFM or LMS.     

Combined administration of G-CSF and GM-CSF stimulates monocyte-derived pro-angiogenic cells in patients with acute myocardial infarction

Mobilization of endothelial progenitor cells has been suggested to contribute to neo-vascularization of ischemic organs. Aim of this study was to investigate whether the combination of granulocyte colony stimulating factor (G-CSF) and granulocyte-macrophage (GM)-CSF may influence the expansion of circulating KDR+ cells in patients with acute myocardial infarction (AMI). KDR+ cells significantly increased in peripheral blood of AMI patients treated with G-CSF and GM-CSF compared to untreated patients. This KDR+ cells population was CD14+ but not CD34+ or CD133+. CD14+/KDR+ cells were also obtained in vitro by culturing mononuclear cells from healthy donors in a Rotary Cell Culture System in the presence of G-CSF + GM-CSF, but not of the individual growth factors. CD14+/KDR+ cells, obtained from patients or from in vitro culture, co-expressed hematopoietic (CD45, CD14) and endothelial markers (CD31, CD105, and VE-cadherin). CD14+/KDR+, but not CD14+/KDR- cells, stimulated the organization of human microvascular endothelial cells into capillary-like structures on Matrigel both in vitro and in vivo. The combination of G-CSF and GM-CSF induced a CD14+/KDR+ cell population with potential pro-angiogenic properties.