Shin-ichi Aota

Requirement for the synergy site for cell adhesion to fibronectin depends on the activation state of integrin α5ß1

We investigated the influence of the activation state of integrin α5β1 on its dependence on the PHSRN synergy site for binding to RGD in fibronectin. K562 and MV3 cells lacked αvβ3 expression and adhered to fibronectin through α5β1. Mel57 cells adhered through αvβ3 and α5β1. A recombinant fibronectin polypeptide, containing five type III repeats from the central cell binding domain 3Fn6-10, and a mutated polypeptide lacking the synergy site were equally effective in promoting Mel57 adhesion. For K562 and MV3, the mutated polypeptide was not or poorly active compared to the control polypeptide. Expression of αvβ3 in MV3 induced strong adhesion to the mutated polypeptide. TS2/16 stimulatory β1-integrin antibodies or Mn induced α5β1-mediated adhesion of K562 and MV3 to GRGDSP. In the presence of TS2/16 or Mn, α5β1-mediated MV3 adhesion to the mutated polypeptide was equally strong as adhesion to the control polypeptide. Mn or TS2/16 induced weak K562 binding to the mutated polypeptide, and in the presence of a combination of phorbol 12-myristate 13-acetate, Mn, and TS2/16, α5β1-mediated K562 adhesion to the mutated and control polypeptide was equally strong. Our findings demonstrate that requirement for the PHSRN synergy site for α5β1-mediated adhesion to RGD in fibronectin depends on the activation state of the integrin.

Pax6 autoregulation mediated by direct interaction of Pax6 protein with the head surface ectoderm-specific enhancer of the mouse Pax6 gene

The Pax6 gene plays crucial roles in eye development and encodes a transcription factor containing both a paired domain and a homeodomain. During embryogenesis, Pax6 is expressed in restricted tissues under the direction of distinct cis-regulatory regions. The head surface ectoderm-specific enhancer of mouse Pax6 directs reporter expression in the derivatives of the ectoderm in the eye, such as lens and cornea, but the molecular mechanism of its control remains largely unknown. We identified a Pax6 protein-responsive element termed LE9 (52 bp in length) within the head surface ectoderm-specific enhancer. LE9, a sequence well conserved across vertebrates, acted as a highly effective enhancer in reporter analyses. Pax6 protein formed in vitro a complex with the distal half of LE9 in a manner dependent on the paired domain. The proximal half of the LE9 sequence contains three plausible sites of HMG domain recognition, and HMG domain-containing transcription factors Sox2 and Sox3 activated LE9 synergistically with Pax6. A scanning mutagenesis experiment indicated that the central site is most important among the three presumptive HMG domain recognition sites. Furthermore, Pax6 and Sox2 proteins formed a complex when they were expressed together. Based on these findings, we propose a model in which Pax6 protein directly and positively regulates its own gene expression, and Sox2 and Sox3 proteins interact with Pax6 protein, resulting in modification of the transcriptional activation by Pax6 protein.

Function and Receptor Specificity of a Minimal 20 Kilodalton Cell Adhesive Fragment of Fibronectin

Previous studies have reached conflicting conclusions about the minimal size and sequences of the fibronectin cell-adhesive domain necessary for retention of high cell adhesive activity. We have expressed a recombinant 20 kDa cell-binding fragment of human fibronectin consisting of the ninth and tenth type III modules, which includes the Arg-Gly-Asp (RGD) cell recognition site and a second cell adhesive domain that acts synergistically with the RGD site. This polypeptide retained a similar activity as a larger 110 kDa fibronectin fragment when used in soluble form in inhibition assays, but it displayed low cell adhesive activity if assayed after direct adsorption to a plastic substrate. However, adhesive function was restored if the fragment was bound to a non-inhibitory anti-fibronectin antibody pre-adsorbed to the plastic substrate. The antibody-bound fragment also promoted cell migration. Both cell spreading and migration were specifically mediated by the alpha 5 beta 1 integrin. Affinity columns containing immobilized 20 kDa cell-binding fragment effectively bound alpha 5-, alpha 3-, and alpha v-containing fibronectin-binding integrins. In contrast, an immobilized 11.5 kDa fragment that contained the RGD sequence but lacked the synergistic sequence was bound only poorly by alpha 5-containing fibronectin receptor integrins, even though the alpha 3- and alpha v-containing integrins bound readily. Our results indicate that the manner in which adhesion proteins are presented to cells is important and that most cell adhesive activity is retained in a minimal 20 kDa segment of fibronectin.

Vinexin Forms a Signaling Complex with Sos and Modulates Epidermal Growth Factor-induced c-Jun N-terminal Kinase/Stress-activated Protein Kinase Activities

Vinexin, a novel protein that plays a key role in cell spreading and cytoskeletal organization, contains three SH3 domains and binds to vinculin through its first and second SH3 domains. We show here that the third SH3 domain binds to Sos, a guanine nucleotide exchange factor for Ras and Rac, both in vitroand in vivo. Point mutations in the third SH3 domain abolished the vinexin-Sos interaction. Stimulation of NIH/3T3 cells with serum, epidermal growth factor (EGF), or platelet-derived growth factor (PDGF) decreased the electrophoretic mobility of Sos and concomitantly inhibited formation of the vinexin-Sos complex. Phosphatase treatment of lysates restored the binding of Sos to vinexin, suggesting that signaling from serum, EGF, or PDGF regulates the vinexin-Sos complex through the Sos phosphorylation. To evaluate the function of vinexin downstream of growth factors, we examined the effects of wild-type and mutant vinexin expression on extracellular signal-regulated kinase (Erk) and c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) activation in response to EGF. Exogenous expression of vinexin β in NIH/3T3 cells enhanced JNK/SAPK activation but did not affect Erk activation. Moreover mutations in the third SH3 domain abolished EGF activation of JNK/SAPK in a dominant-negative fashion, whereas they slightly stimulated Erk. Together these results suggest that vinexin can selectively modulate EGF-induced signal transduction pathways leading to JNK/SAPK kinase activation.

The ActR-I activin receptor protein is expressed in notochord, lens placode and pituitary primordium cells in the mouse embryo

ActR-I is a type I serine/threonine kinase receptor which has been shown to bind activin and bone morphogenetic proteins (BMPs). To study the function of ActR-I, we have generated novel monoclonal antibodies that specifically recognize the extracellular domain of mouse ActR-I. We examined the level of ActR-I protein during mouse development by immunohistochemistry. We found that in the embryonic body, ActR-I protein first appears in a restricted part of the primitive streak region and is present throughout the length of notochord. Furthermore, ActR-I protein is expressed in the facial sensory organ primordia, including eye area, otic vesicle and olfactory placode, which all contain invaginating ectoderm. In addition, ActR-I is produced in pituitary primordium (Rathkes pouch), mammary buds and the epithelial layer of branchial arches. Interestingly, in the lens placodes and in early Rathkes pouch, ActR-I protein is transiently localized at the apical surface of the epithelial cells, indicating the presence of an apical-basal asymmetry in these cells.

Smad7 induces G0/G1 cell cycle arrest in mesenchymal cells by inhibiting the expression of G1 cyclins

The major Smad pathways serve in regulating the expression of genes downstream of TGFβ signals. In this study, we examined the effects of sustained Smad7 expression in cultured cells. Interestingly, Smad7 caused various mesenchymal cells, including NIH3T3 fibroblast and ST2 bone‐marrow stromal cells, to undergo a marked morphological alteration into a flattened cell shape, but kept them alive for as long as 60 days. Furthermore, Smad7 arrested the proliferation of the cells even before they reached confluence. These cells became quiescent in G0/G1 phase and accumulated a hypophosphorylated form of retinoblastoma. The cytostatic effect of Smad7 was closely associated with a preceding decrease in the levels of G1 cyclins, such as cyclin D1 and cyclin E. Accordingly, ectopic cyclin E was able to overcome the Smad7‐induced arrest of proliferation. These results indicate that Smad7 functions upstream of G1 cyclins and suggest a novel role for Smad7 as an antiproliferative factor. In contrast to the growth of mesenchymal cells, that of epithelial cells was little susceptible to Smad7. The present findings raise the possibility that a link between Smad7 and the G1 to S phase transition may also contribute to the cell cycle control by certain Smad7‐inducing stimuli in a cell‐type‐dependent fashion.

Expression of fibronection fragments inE. coli

Methods are described for high-efficiency expression of fibronectin fragments by using anE. coli protein expression system. Complementary DNA (cDNA) fragments cloned in a T7 promoter-based expression plasmid were expressed inE. coli strain BL21(DE3, pLysS). The resulting fibronectin fragments were purified by DEAE ion-exchange chromatography. This method can produce a large quantity of protein fragments for various studies including functional assays and biophysical measurements.