lucifer-yellow and lactacystin

Gap junctions within extracellular matrix (ECM)-defined boundaries ensure synchronous activity between cells destined to become functional mediators that regulate cell behavior. However, the role of ECM in connexin (Cx) function in mouse embryonic stem cells (mESCs) has not been elucidated. Therefore, we examined the role of laminin-111 in the control of Cx43 functions and related signal pathways in mESCs. ECM components (laminin-111, fibronectin, and collagen I) increased Cx43 phosphorylation and decreased Lucifer yellow (Ly) diffusion. In addition, laminin-111 increased the proliferation index through reduction of gap junctional intercellular communication (GJIC), which was confirmed by 18α-glycyrrhetinic acid (18α-GA). Laminin-111 increased phosphorylation of focal adhesion kinase (FAK)/Src and protein kinase C (PKC), which were inhibited by integrin β1 antibody (Ab) and laminin receptor-1 (LR-1) Ab, respectively. In addition, inhibition of both FAK/Src and PKC blocked Cx43 phosphorylation. Laminin-111 increased the Ras homolog gene family, member A (RhoA) activation, which was blocked by FAK/Src and PKC inhibitors, suggesting the existence of parallel pathways that merge at RhoA. Inhibition of RhoA reversed the laminin-111-induced increase of Cx43 phosphorylation and reduction of GJIC. Laminin-111 also stimulated the dissociation of Cx43/ZO-1 complex followed by disruption of Cx43/drebrin and Cx43/F-actin complexes, which were reversed by C3 (RhoA inhibitor). ZO-1 small interfering (si) RNA significantly decreased Ly diffusion. Moreover, laminin-111 decreased Cx43 labeling at the intercellular junction, whereas pretreatment with degradation inhibitors (lysosomal protease inhibitor, chloroquine; proteasome inhibitor, lactacystin) increased Cx43 expression, reversely. In conclusion, laminin-111 stimulated mESC proliferation through a reduction of GJIC via RhoA-mediated Cx43 phosphorylation and Cx43/ZO-1/drebrin complex instability-mediated Cx43 degradation.

Topics: Acetylcysteine; Actins; Animals; Cell Proliferation; Chloroquine; Connexin 43; Embryonic Stem Cells; Extracellular Matrix; Fibronectins; Focal Adhesion Kinase 1; Gap Junctions; Glycyrrhetinic Acid; Isoquinolines; Laminin; Mice; Multiprotein Complexes; Neuropeptides; Phosphorylation; Protease Inhibitors; Protein Kinase C; Protein Stability; Proteolysis; Receptors, Laminin; rho GTP-Binding Proteins; rhoA GTP-Binding Protein; RNA, Small Interfering; Zonula Occludens-1 Protein

Deposition of amyloid beta protein in the brain is the major pathological feature of Alzheimer's disease. Amyloid beta protein is generated from beta-amyloid precursor protein by beta-secretase and gamma-secretase. Proteolytic processing of amyloid precursor protein at the beta site by BACE1 is essential to generate amyloid beta protein. BACE1, the major beta-secretase involved in cleaving amyloid precursor protein, has been identified as a type 1 membrane-associated aspartyl protease. In this study, we found that BACE1 gene expression is controlled by a TATA-less promoter. BACE1 gene expression is tightly regulated at the transcriptional level and the transcription factor Sp1 plays an important role in regulation of BACE1 to process amyloid precursor protein generating amyloid beta protein. Furthermore, we found that BACE1 protein is ubiquitinated, and the degradation of BACE1 proteins and amyloid precursor protein processing are regulated by the ubiquitin-proteasome pathway.

Topics: Acetylcysteine; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Analysis of Variance; Aspartic Acid Endopeptidases; Cell Line, Tumor; Cloning, Molecular; Cysteine Proteinase Inhibitors; Endopeptidases; Enzyme-Linked Immunosorbent Assay; Fluorescent Antibody Technique; Gene Expression Regulation; Humans; Isoquinolines; Mutagenesis; Peptide Fragments; Peptide Hydrolases; Phosphopyruvate Hydratase; Promoter Regions, Genetic; Sp1 Transcription Factor; Time Factors; Transfection