Rapid loss of microvascular integrin expression during focal brain ischemia reflects neuron injury.

The integrity of cerebral microvessels requires the close apposition of the endothelium to the astrocyte endfeet. Integrins alpha1beta1 and alpha6beta4 are cellular matrix receptors that may contribute to cerebral microvascular integrity. It has been hypothesized that focal ischemia alters integrin expression in a characteristic time-dependent manner consistent with neuron injury. The effects of middle cerebral artery occlusion (MCAO) and various periods of reperfusion on microvasclar integrin alpha1beta1 and alpha6beta4 expression were examined in the basal ganglia of 17 primates. Integrin subunits alpha1 and beta1 colocalized with the endothelial cell antigen CD31 in nonischemic microvessels and with glial fibrillary acidic protein on astrocyte fibers. Rapid, simultaneous, and significant disappearance of both integrin alpha1 and beta1 subunits and integrin alpha6beta4 occurred by 2 hours MCAO, which was greatest in the region of neuron injury (ischemic core, Ic), and progressively less in the peripheral (Ip) and nonischemic regions (N). Transcription of subunit beta1 mRNA on microvessels increased significantly in the Ic/Ip border and in multiple circular subregions within Ic. Microvascular integrin alpha1beta1 and integrin alpha6beta4 expression are rapidly and coordinately lost in Ic after MCAO. With loss of integrin alpha1beta1, multiple regions of microvascular beta1 mRNA up-regulation within Ic suggest that microvessel responses to focal ischemia are dynamic, and that multiple cores, not a single core, are generated. These changes imply that microvascular integrity is modified in a heterogeneous, but ordered pattern.

Divalent cations regulate the organization of integrins alpha v beta 3 and alpha v beta 5 on the cell surface.

Extracellular divalent cations are important regulators of integrin ligand binding activity. In this study we evaluated how divalent cations affect the organization of integrins into focal adhesion sites. Integrins alpha v beta 3 and alpha v beta 5 were compared because they share a high degree of structural homology and because both integrins mediate cell adhesion to vitronectin. On MG-63 osteosarcoma cells, we found that both the extent and pattern of integrin organization was regulated by the type of extracellular divalent ion. Integrin alpha v beta 3 organized in focal contacts when Mn2+ or Mg2+ was present, but not in Ca2+. In contrast, alpha v beta 5 organized in focal contacts only when Ca2+ or Mg2+ was present. Integrin alpha v beta 5 clustered in a centrally located punctate field on the ventral surface of the cell in the presence of Mn2+. These observations reveal a previously unappreciated role for divalent ions in regulating the organization of integrins into focal adhesion sites.

The phorbol ester TPA regulates collagen gene expression at the transcriptional level.

Previously, we reported that growth activation of quiescent 3T3-L1 cells by TPA led to a rapid increase of pro-alpha 2 (I) collagen mRNA and protein, while induction of pro-alpha 2 (I) was not observed in VT-1 cells, a line non-mitogenic in the presence of TPA (26). Here, we further examine the expression of pro-alpha 2 (I) collagen during mitogenic stimulation at the molecular level. In addition to pro-alpha 2 (I) mRNA, TPA treatment increased mRNA production of other collagen family members, pro-alpha 1 (I) and pro-alpha 1 (III) although in reduced amounts relative to pro-alpha 2 (I). In contrast to pro-alpha 2 (I), the mRNA expression profiles of several protooncogenes were regulated in both VT-1 and 3T3-L1 cells. Consistent with increased mRNA levels, TPA treated 3T3-L1 cells produced a matrix abundant in collagen type I protein. In vitro nuclear "run-on" transcription assays demonstrated a 4-fold increase in pro-alpha 2 (I) mRNA that was maximal within 10 min of TPA treatment. Using a chloramphenicol-acetyl transferase (CAT) assay, we identified a TPA sensitive domain within the promoter of the COL1A2 gene. These results establish COL1A2 as an early growth responsive gene, and that its regulation is PKC dependent. Additionally, the increased expression of protooncogenes and transin during TPA stimulation of non-mitogenic VT-1 cells indicated that the regulation of these genes is independent of PKC, indicating the existence of multiple regulatory mechanisms amongst early response genes.

Characterization of monoclonal antibodies against integrin alpha V beta 5.

A panel of monoclonal antibodies directed against the human integrin alpha V beta 5 has been generated. Mice were immunized using human alpha V beta 5 purified from placenta. Two hundred hybridoma clones were screened by ELISA and 14 were found to react with purified integrin alpha V beta 5. Here we characterize the monoclonal antibodies secreted by three clones, 14H4, 6B9, and 15F11. Two of these antibodies, 6B9 and 15F11, are highly specific for integrin alpha V beta 5. These two antibodies do not bind the highly homologous integrin alpha V beta 3. The binding of 6B9 and 15F11 to integrin alpha V beta 5 is not influenced by ligand nor by divalent cation. Antibody 14H4, obtained in the same fusion, recognizes the alpha v subunit.

Regulation of integrin function and cellular adhesion.

The integrins are a family of adhesion receptors involved in many physiological functions. These molecules are characterized by an ability to dynamically regulate their ligand binding affinity. Several integrins become "activated" or achieve the high affinity state in response to extracellular agonists or signals. High affinity ligand binding does not result from an increase in receptor number or from changes in the receptor microenvironment. Rather, evidence suggests these altered affinity states result from the varied conformations of these molecules. Understanding how these conformational changes are achieved remains an area of great interest in the field. In this review, we will discuss several means and potential mechanisms of integrin activation. First, we will focus on "activators" such as antibodies, peptides, and cations. For the most part, these agents can be viewed as nonphysiological activators that directly effect integrin conformational changes. Later we will discuss how conformational changes are achieved in a physiological sense. Many physiological activators stimulate signal transduction pathways inside the cell and are believed to transmit these signals outward to effect conformational changes. An understanding of integrin activation mechanisms is important as it might suggest ways to regulate cell adhesion in pathology and disease.

Phorbol-ester-mediated expression of the collagen type I pro-alpha 2 gene in mouse 3T3-L1 cells and its absence in a phorbol 12-myristate 13-acetate-non-responsive variant.

We have previously isolated a phorbol 12-myristate 13-acetate (PMA)-non-responsive variant line (VT-1) from mouse 3T3-L1 cells [Shimizu, Fujiki, Sugimura & Shimizu (1986) Cancer Res. 46, 4027-4031]. Differential hybridization of cDNAs obtained from PMA-treated 3T3-L1 and VT-1 cells resulted in the isolation of a number of unique cDNA clones, including one with a high degree of sequence similarity to the type I pro-alpha 2 collagen gene (COL1A2) [Amagai, Inokuchi, Nishikawa, Shimizu & Shimizu (1989) Somat. Cell Mol. Genet. 15, 153-158]. Here we examined the expression of type I collagen pro-alpha 2 [pro-alpha 2(I)] mRNA and production of type I collagen in these two cell lines. In quiescent cells, the pro-alpha 2(I) steady-state mRNA levels were four times greater in 3T3-L1 cells than in VT-1 cells. PMA addition caused the steady-state levels of pro-alpha 2(I) mRNA to be six times greater in 3T3-L1 cells than in VT-1 cells, with a maximum at 30-60 min. The pro-alpha 2(I) protein levels in the extracellular matrix or culture media of 3T3-L1 cells were substantially elevated by PMA treatment, but no significant increase was detected in VT-1 cells. The correlation of collagen expression with a PMA-mediated mitogenic response suggests a new role for collagen as an early component of mitogenic signal transduction.

Regulation of glycerol uptake by the phosphoenolpyruvate-sugar phosphotransferase system in Bacillus subtilis.

Enteric bacteria have been previously shown to regulate the uptake of certain carbohydrates (lactose, maltose, and glycerol) by an allosteric mechanism involving the catalytic activities of the phosphoenolpyruvate-sugar phosphotransferase system. In the present studies, a ptsI mutant of Bacillus subtilis, possessing a thermosensitive enzyme I of the phosphotransferase system, was used to gain evidence for a similar regulatory mechanism in a gram-positive bacterium. Thermoinactivation of enzyme I resulted in the loss of methyl alpha-glucoside uptake activity and enhanced sensitivity of glycerol uptake to inhibition by sugar substrates of the phosphotransferase system. The concentration of the inhibiting sugar which half maximally blocked glycerol uptake was directly related to residual enzyme I activity. Each sugar substrate of the phosphotransferase system inhibited glycerol uptake provided that the enzyme II specific for that sugar was induced to a sufficiently high level. The results support the conclusion that the phosphotransferase system regulates glycerol uptake in B. subtilis and perhaps in other gram-positive bacteria.