Scribble, Lgl1, and myosin IIA interact with α-/β-catenin to maintain epithelial junction integrity

ABSTRACTE-cadherin-catenin complex together with the cytoskeleton, builds the core of Adherens junctions (AJs). It has been reported that Scribble stabilizes the coupling of E-cadherin with catenins promoting epithelial cell adhesion, but the mechanism remains unknown. We show that Scribble, Lgl1, and NMII-A reside in a complex with E-cadherin-catenin complex. Depletion of either Scribble or Lgl1 disrupts the localization of E-cadherin-catenin complex to AJs. aPKCζ phosphorylation of Lgl1 regulates AJ localization of Lgl1 and E-cadherin-catenin complexes. Both Scribble and Lgl1 regulate the activation and recruitment of NMII-A at AJs. Finally, Scribble and Lgl1 are downregulated by TGFβ-induced EMT, and their re-expression during EMT impedes its progression. Our results provide insight into the mechanism regulating AJ integrity by Scribble, Lgl1, and NMII-A

aPKCζ affects directed cell migration through the regulation of myosin light chain phosphorylation

<p>Cell motility is an essential cellular process for a variety of biological events. It requires cross-talk between the signaling and the cytoskeletal systems. Despite the recognized importance of aPKCζ for cell motility, there is little understanding of the mechanism by which aPKCζ mediates extracellular signals to the cytoskeleton. In the present study, we report that aPKCζ is required for the cellular organization of acto-non-muscle myosin II (NMII) cytoskeleton, for proper cell adhesion and directed cell migration. We show that aPKCζ mediates EGF-dependent RhoA activation and recruitment to the cell membrane. We also show that aPKCζ mediates EGF-dependent myosin light chain (MRLC) phosphorylation that is carried out by Rho-associated protein kinase (ROCK), and that aPKCζ is required for EGF-dependent phosphorylation and inhibition of the myosin phosphatase targeting subunit (MYPT). Finally, we show that aPKCζ mediates the spatial organization of the acto-NMII cytoskeleton in response to EGF stimulation. Our data suggest that aPKCζ is an essential component regulator of acto-NMII cytoskeleton organization leading to directed cell migration, and is a mediator of the EGF signal to the cytoskeleton.</p

Scribble, Lgl1, and myosin IIA interact with α-/β-catenin to maintain epithelial junction integrity

E-cadherin-catenin complex together with the cytoskeleton, builds the core of Adherens junctions (AJs). It has been reported that Scribble stabilizes the coupling of E-cadherin with catenins promoting epithelial cell adhesion, but the mechanism remains unknown. We show that Scribble, Lgl1, and NMII-A reside in a complex with E-cadherin-catenin complex. Depletion of either Scribble or Lgl1 disrupts the localization of E-cadherin-catenin complex to AJs. aPKCζ phosphorylation of Lgl1 regulates AJ localization of Lgl1 and E-cadherin-catenin complexes. Both Scribble and Lgl1 regulate the activation and recruitment of NMII-A at AJs. Finally, Scribble and Lgl1 are downregulated by TGFβ-induced EMT, and their re-expression during EMT impedes its progression. Our results provide insight into the mechanism regulating AJ integrity by Scribble, Lgl1, and NMII-A.</p

Trans-Binding Mechanism of Ubiquitin-like Protein Activation Revealed by a UBA5-UFM1 Complex

Modification of proteins by ubiquitin or ubiquitin-like proteins (UBLs) is a critical cellular process implicated in a variety of cellular states and outcomes. A prerequisite for target protein modification by a UBL is the activation of the latter by activating enzymes (E1s). Here, we present the crystal structure of the non-canonical homodimeric E1, UBA5, in complex with its cognate UBL, UFM1, and supporting biochemical experiments. We find that UBA5 binds to UFM1 via a trans-binding mechanism in which UFM1 interacts with distinct sites in both subunits of the UBA5 dimer. This binding mechanism requires a region C-terminal to the adenylation domain that brings UFM1 to the active site of the adjacent UBA5 subunit. We also find that transfer of UFM1 from UBA5 to the E2, UFC1, occurs via a trans mechanism, thereby requiring a homodimer of UBA5. These findings explicitly elucidate the role of UBA5 dimerization in UFM1 activation

The Sirt1 Activators SRT2183 and SRT3025 Inhibit RANKL-Induced Osteoclastogenesis in Bone Marrow-Derived Macrophages and Down-Regulate Sirt3 in Sirt1 Null Cells.

Increased osteoclast-mediated bone resorption is characteristic of osteoporosis, malignant bone disease and inflammatory arthritis. Targeted deletion of Sirtuin1 (Sirt1), a key player in aging and metabolism, in osteoclasts results in increased osteoclast-mediated bone resorption in vivo, making it a potential novel therapeutic target to block bone resorption. Sirt1 activating compounds (STACs) were generated and were investigated in animal disease models and in humans however their mechanism of action was a source of controversy. We studied the effect of SRT2183 and SRT3025 on osteoclastogenesis in bone-marrow derived macrophages (BMMs) in vitro, and discovered that these STACs inhibit RANKL-induced osteoclast differentiation, fusion and resorptive capacity without affecting osteoclast survival. SRT2183 and SRT3025 activated AMPK, increased Sirt1 expression and decreased RelA/p65 lysine310 acetylation, critical for NF-κB activation, and an established Sirt1 target. However, inhibition of osteoclastogenesis by these STACs was also observed in BMMs derived from sirt1 knock out (sirt1-/-) mice lacking the Sirt1 protein, in which neither AMPK nor RelA/p65 lysine 310 acetylation was affected, confirming that these effects require Sirt1, but suggesting that Sirt1 is not essential for inhibition of osteoclastogenesis by these STACs under these conditions. In sirt1 null osteoclasts treated with SRT2183 or SRT3025 Sirt3 was found to be down-regulated. Our findings suggest that SRT2183 and SRT3025 activate Sirt1 and inhibit RANKL-induced osteoclastogenesis in vitro however under conditions of Sirt1 deficiency can affect Sirt3. As aging is associated with reduced Sirt1 level and activity, the influence of STACs on Sirt3 needs to be investigated in vivo in animal and human disease models of aging and osteoporosis

Overexpression of UBA5 in Cells Mimics the Phenotype of Cells Lacking UBA5

Ufmylation is a posttranslational modification in which the modifier UFM1 is attached to target proteins. This conjugation requires the concerted work of three enzymes named UBA5, UFC1, and UFL1. Initially, UBA5 activates UFM1 in a process that ends with UFM1 attached to UBA5’s active site Cys. Then, in a trans-thiolation reaction, UFM1 is transferred from UBA5 to UFC1, forming a thioester bond with the latter. Finally, with the help of UFL1, UFM1 is transferred to the final destination—a lysine residue on a target protein. Therefore, not surprisingly, deletion of one of these enzymes abrogates the conjugation process. However, how overexpression of these enzymes affects this process is not yet clear. Here we found, unexpectedly, that overexpression of UBA5, but not UFC1, damages the ability of cells to migrate, in a similar way to cells lacking UBA5 or UFC1. At the mechanistic level, we found that overexpression of UBA5 reverses the trans-thiolation reaction, thereby leading to a back transfer of UFM1 from UFC1 to UBA5. This, as seen in cells lacking UBA5, reduces the level of charged UFC1 and therefore harms the conjugation process. In contrast, co-expression of UBA5 with UFM1 abolishes this effect, suggesting that the reverse transfer of UFM1 from UFC1 to UBA5 depends on the level of free UFM1. Overall, our results propose that the cellular expression level of the UFM1 conjugation enzymes has to be tightly regulated to ensure the proper directionality of UFM1 transfer

Sirt1 Promotes a Thermogenic Gene Program in Bone Marrow Adipocytes: From Mice to (Wo)Men

Bone marrow adipose tissue (MAT) is influenced by nutritional cues, and participates in whole body energy metabolism. To investigate the role of Sirtuin1 (Sirt1), a key player in metabolism, in MAT, marrow adiposity was evaluated in inbred 5-month-old 129/Sv Sirt1 haplo-insufficient (Sirt1Δ/+) and wild type (WT) mice. Decreased expression of the thermogenic genes: Prdm16, Pgc1α, Foxc2, Dio2, and β3AR was detected in whole tibiae derived from Sirt1Δ/+ compared to WT female mice. Similarly, decreased expression of Prdm16 and Pgc1α was observed in primary bone marrow mesenchymal stem cell (BM-MSC) cultures obtained from Sirt1Δ/+ compared to WT female mice, suggesting a cell autonomous effect of Sirt1 in BM-MSCs. In vitro, Sirt1 over-expression in the mesenchymal embryonic fibroblast stem cell line C3HT101/2 increased Pgc1α and Prdm16 protein level. Similarly, pharmacologic activation of Sirt1 by SRT3025 increased Foxc2, Pgc1α, Dio2, Tfam, and Cyc1 expression while inhibition of Sirt1 by EX527 down-regulated UCP1 in C3HT101/2 cells. Importantly, in human femoral BM-MSCs obtained from female patients undergoing hip operations for fracture or osteoarthritis, Sirt1 activation by SRT3025 increased PGC1α mRNA and protein level. Blocking sclerostin, an inhibitor of the WNT pathway and a Sirt1 target, by the monoclonal humanized antibody (Sc-AbII), stimulated β3AR, PRDM16, and UCP1 gene expression, and increased PGC1α protein level. These results show that Sirt1 stimulates a thermogenic gene program in marrow adipocytes in mice and humans via PGC1α activation and sclerostin inhibition. The implications of these findings to bone health, hematopoiesis and whole body energy metabolism remain to be investigated.ISSN:1664-239

SRT2183 inhibits RANKL-induced osteoclastogenesis and pit formation in s<i>irt1</i>^<i>-/-</i> BMMs.

<p>(A) Sirt1 expression in WT- and in <i>Sirt1</i>^<i>-/-</i>-derived osteoclasts. PCR amplification of exons 1–9 of the s<i>irt1</i> gene (left panel) and Western blot analysis with Sirt1 antibody (right panel) demonstrates complete loss of Sirt1 protein in osteoclasts obtained from <i>Sirt1</i>^<i>Δ/Δ</i> (<i>Sirt1</i>^<i>-/-</i>) mice. (B) The effect of SRT2183 on osteoclast differentiation in <i>Sirt1</i>^<i>-/-</i>-derived BMMs. BMMs were inducted to osteoclastogenesis with RANKL in the presence or absence of SRT2183. TRAP staining performed 4 days post induction. (C) The effect of SRT2183 on pit formation in <i>Sirt1</i>^<i>-/-</i>-derived BMMs stimulated with RANKL. An eroded area (left panel) and pit formation assay (right) are shown. (D) The effect of SRT2183 on p65 acetylation (Lys310). Western blot analysis of p65K310 ac and p65 in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. (E) The effect of SRT2183 on AMPKα phosphorylation (Thr172). Western blot analysis of pAMPKα and AMPKα in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. (F) The effect of SRT2183 on IκBα protein level. Western blot analysis of IκBα and GAPDH in SRT2183- and vehicle-treated BMMs 24 hours post RANKL stimulation. (G-H) The effect of SRT2183 on Sirt3 protein (G) and gene expression (H). Western blot analysis of Sirt3 and GAPDH in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation (G). Gene expression analysis by quantitative Real-Time PCR 4 days post RANKL stimulation is shown. Results are relative to <i>Polr2a</i> (H). (I) The effect of SRT2183 on superoxide dismutase 2 (Sod2) Lys68 acetylation. Western blot analysis of acetylated (ac) Sod2K68 and Sod2 in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. Data are Mean ± SEM (n = 3 independent experiments), analyzed by paired Student's <i>t</i>-test paired (C) or one-sample Student's <i>t</i>-test (H-I); ***<i>P</i><0.001, ****<i>P</i><0.0001, versus vehicle-treated BMMs. Magnification X40; scale bar 1mm.</p

SRT2183 inhibits RANKL-induced osteoclastogenesis in bone marrow-derived macrophages (BMMs).

<p>(A) Chemical structure of SRT2183. (B-C) TRAP staining of SRT2183 or vehicle-treated BMMs inducted to osteoclastogenesis 4 days post RANKL stimulation (B). Total number of osteoclasts (left panel) and number of multinuclear cells (right) are shown (C). (D) The effect of SRT2183 on pit formation by RANKL-induced BMMs. A pit formation assay (left panel) and eroded area (right) are shown. (E) Time course of the effect of SRT2183 or vehicle (DMSO) administration on osteoclast differentiation. SRT2183 or a vehicle was added in the proliferation (a), differentiation (b, c) or maturation (c, d) phase. Arrows indicate periods of treatments with SRT2183. TRAP staining is shown. (F) The effect of SRT2183 on cell proliferation during the proliferation phase. SRT2183 or a vehicle were co-administrated with M-CSF for 72 hours on day of plating. (G-H) The effect of SRT2183 on cell viability and apoptosis during the proliferation phase (G) and the differentiation and maturation phase (H). SRT2183 or vehicle were co-administrated with M-CSF for 72 hours on day of plating (G) or with RANKL 3 days post plating (H). The graphs illustrate fold change in Caspase 3 activity and the percent change in living cells with time. Data are Mean ±SEM (n = 3 independent experiments), analyzed by 2 way ANOVA with nuclei number and treatment as the independent variables followed by Sidak's post-hoc correction (C), paired Student's <i>t</i>-test (D), one-sample Student's <i>t</i>-test (F-H), *<i>P</i><0.05; **<i>P</i><0.01 compared to vehicle-treated BMMs. Magnification X40; Scale bar 1mm.</p