Mammary epithelial cell phagocytosis downstream of TGF-β3 is characterized by adherens junction reorganization

After weaning, during mammary gland involution, milk-producing mammary epithelial cells undergo apoptosis. Effective clearance of these dying cells is essential, as persistent apoptotic cells have a negative impact on gland homeostasis, future lactation and cancer susceptibility. In mice, apoptotic cells are cleared by the neighboring epithelium, yet little is known about how mammary epithelial cells become phagocytic or whether this function is conserved between species. Here we use a rat model of weaning-induced involution and involuting breast tissue from women, to demonstrate apoptotic cells within luminal epithelial cells and epithelial expression of the scavenger mannose receptor, suggesting conservation of phagocytosis by epithelial cells. In the rat, epithelial transforming growth factor-β (TGF-β) signaling is increased during involution, a pathway known to promote phagocytic capability. To test whether TGF-β enhances the phagocytic ability of mammary epithelial cells, non-transformed murine mammary epithelial EpH4 cells were cultured to achieve tight junction impermeability, such as occurs during lactation. TGF-β3 treatment promoted loss of tight junction impermeability, reorganization and cleavage of the adherens junction protein E-cadherin (E-cad), and phagocytosis. Phagocytosis correlated with junction disruption, suggesting junction reorganization is necessary for phagocytosis by epithelial cells. Supporting this hypothesis, epithelial cell E-cad reorganization and cleavage were observed in rat and human involuting mammary glands. Further, in the rat, E-cad cleavage correlated with increased γ-secretase activity and β-catenin nuclear localization. In vitro, pharmacologic inhibitors of γ-secretase or β-catenin reduced the effect of TGF-β3 on phagocytosis to near baseline levels. However, β-catenin signaling through LiCl treatment did not enhance phagocytic capacity, suggesting a model in which both reorganization of cell junctions and β-catenin signaling contribute to phagocytosis downstream of TGF-β3. Our data provide insight into how mammary epithelial cells contribute to apoptotic cell clearance, and in light of the negative consequences of impaired apoptotic cell clearance during involution, may shed light on involution-associated breast pathologies.Effective clearance of apoptotic cells is important in maintaining tissue homeostasis. Weaning-induced mammary gland involution is a unique model for studying apoptotic cell clearance, as 80–90% of the milk-producing mammary epithelium undergoes apoptosis to return the gland to a non-secretory state.¹ Professional phagocytes, such as macrophages, are recruited into the involuting mammary gland; however, they are thought to have a limited role in the clearance of dying secretory cells, as in mice, peak macrophage infiltration occurs after the majority of apoptotic cell removal.² Rather, the neighboring mammary epithelial cells themselves appear to be the primary cell type responsible for apoptotic cell clearance during involution.² Rapid and efficient apoptotic cell clearance is essential, as persistence of apoptotic cells can result in the release of cell fragments into the local environment and subsequent autoimmunity.³ Importantly, impaired apoptotic cell clearance in the postpartum mammary gland results in local inflammation, fibrosis and epithelial cell hyperplasia.^{4, 5}Although there is increasing evidence that phagocytosis by mammary epithelial cells has a crucial role in maintaining tissue homeostasis in the involuting murine mammary gland, little is known about how mammary epithelial cells become phagocytic during postpartum involution. One of the key changes in the mammary epithelium that may contribute to acquisition of a phagocytic phenotype is reorganization of epithelial cell junctions. During lactation, tight junctions between mammary epithelial cells become highly impermeable, which assures localization of milk within the mammary ducts.⁶ With weaning, this impermeability is lost,⁶ consistent with tight junction reorganization. Furthermore, reorganization of adherens junctions is also observed upon the switch from lactation to involution.⁷ Given that professional phagocytes such as macrophages do not exist in monolayers with cell cell junctions, disruption of epithelial cell junctions at the onset of mammary gland involution may be required for mammary epithelial cells to become phagocytic.One candidate cytokine for promoting epithelial cell junction reorganization and phagocytosis is transforming growth factor-β (TGF-β). Binding of TGF-β to the TGF-β type II receptor (TβRII) activates canonical signaling through a signaling cascade involving the TGF-β type I receptor, receptor-associated Smads (Smad2/3) and Smad4. TGF-β protein and mRNA levels are significantly increased in the mammary gland on the switch from lactation to involution, with increased expression persisting through at least 9 days post weaning.^{8, 9} Of the three TGF-β isoforms (TGF-β1, -β2 and -β3), TGF-β3 increases the greatest upon the lactation-to-involution switch.^{8, 9, 10, 11} Overexpressing TGF-β3 or depleting Smad3 or TβRII in the mammary epithelium reveals a necessary role for TGF-β in promoting apoptosis early during involution.^{10, 12, 13, 14} However, sustained TGF-β expression throughout the postpartum involution window suggests additional roles for TGF-β that extend beyond apoptosis induction, including influencing extracellular matrix remodeling and immune cell composition.^{8, 10, 12, 13, 14, 15} TGF-β is known to increase the phagocytic capacity of retinal pigment epithelial cells, fibroblasts and macrophages,^{16, 17, 18} although a role for TGF-β in mediating apoptotic cell clearance by phagocytic mammary epithelial cells has not been explored. Furthermore, TGF-β is implicated in tight junction disruption in the mammary gland and has known roles in adherens junction disassembly, making it an intriguing target to investigate in the promotion of a phagocytic phenotype in mammary epithelial cells.^{6, 19}Currently, it is unknown whether the mammary epithelium has a role in apoptotic cell clearance in species other than mice. Therefore, we evaluated rat and human involution mammary tissue for apoptotic cell clearance by the mammary epithelium. Further, as addressing the role of TGF-β in promoting phagocytosis by mammary epithelial cells during gland involution is challenging due to impaired cell death in the absence of TGF-β signaling,^{12, 13, 14} we developed an in vitro model to investigate the role of TGF-β3 in mammary epithelial cell junction reorganization and phagocytosis.We demonstrate engulfment of apoptotic cells by mammary epithelial cells during weaning-induced involution in both rats and women, supportive of phagocytosis being a conserved feature of mammary epithelium during postpartum involution. Using our murine mammary epithelial culture model that mimics the high junctional resistance of the lactating gland, we show that TGF-β3 promotes phagocytic capability and identify a potential role for cell–cell junction disruption in epithelial cell phagocytosis. Furthermore, we identify a previously unreported role for the intramembrane protease γ-secretase in the promotion of phagocytosis by TGF-β3. In light of the negative consequences of impaired apoptotic cell clearance during postpartum involution,^{4, 5} our data provide insight into how mammary epithelial cells may contribute to apoptotic cell clearance during this time.     

β-actin and γ-actin are each dispensable for auditory hair cell development but required for Stereocilia maintenance

Hair cell stereocilia structure depends on actin filaments composed of cytoplasmic β-actin and γ-actin isoforms. Mutations in either gene can lead to progressive hearing loss in humans. Since β-actin and γ-actin isoforms are 99% identical at the protein level, it is unclear whether each isoform has distinct cellular roles. Here, we compared the functions of β-actin and γ-actin in stereocilia formation and maintenance by generating mice conditionally knocked out for Actb or Actg1 in hair cells. We found that, although cytoplasmic actin is necessary, neither β-actin nor γ-actin is required for normal stereocilia development or auditory function in young animals. However, aging mice with β-actin- or γ-actin-deficient hair cells develop different patterns of progressive hearing loss and distinct pathogenic changes in stereocilia morphology, despite colocalization of the actin isoforms. These results demonstrate overlapping developmental roles but unique post-developmental functions for β-actin and γ-actin in maintaining hair cell stereocilia.     

The tyrosine phosphatase SHP2 regulates recovery of endothelial adherens junctions through control of β-catenin phosphorylation

Impaired endothelial barrier function results in a persistent increase in endothelial permeability and vascular leakage. Repair of a dysfunctional endothelial barrier requires controlled restoration of adherens junctions, comprising vascular endothelial (VE)-cadherin and associated β-, γ-, α-, and p120-catenins. Little is known about the mechanisms by which recovery of VE-cadherin–mediated cell–cell junctions is regulated. Using the inflammatory mediator thrombin, we demonstrate an important role for the Src homology 2-domain containing tyrosine phosphatase (SHP2) in mediating recovery of the VE-cadherin–controlled endothelial barrier. Using SHP2 substrate-trapping mutants and an in vitro phosphatase activity assay, we validate β-catenin as a bona fide SHP2 substrate. SHP2 silencing and SHP2 inhibition both result in delayed recovery of endothelial barrier function after thrombin stimulation. Moreover, on thrombin challenge, we find prolonged elevation in tyrosine phosphorylation levels of VE-cadherin–associated β-catenin in SHP2-depleted cells. No disassembly of the VE-cadherin complex is observed throughout the thrombin response. Using fluorescence recovery after photobleaching, we show that loss of SHP2 reduces the mobility of VE-cadherin at recovered cell–cell junctions. In conclusion, our data show that the SHP2 phosphatase plays an important role in the recovery of disrupted endothelial cell–cell junctions by dephosphorylating VE-cadherin–associated β-catenin and promoting the mobility of VE-cadherin at the plasma membrane.     

The bacterial outer membrane β-barrel assembly machinery

β-Barrel proteins found in the outer membrane of Gram-negative bacteria serve a variety of cellular functions. Proper folding and assembly of these proteins are essential for the viability of bacteria and can also play an important role in virulence. The β-barrel assembly machinery (BAM) complex, which is responsible for the proper assembly of β-barrels into the outer membrane of Gram-negative bacteria, has been the focus of many recent studies. This review summarizes the significant progress that has been made toward understanding the structure and function of the bacterial BAM complex.     

Odontoblast β-catenin signaling regulates fenestration of mouse Hertwig's epithelial root sheath

The interaction between Hertwig's epithelial root sheath(HERS) and the adjacent mesenchyme is vitally important in mouse tooth root development. We previously generated odontoblast-specific Ctnnb1(encoding β-catenin) deletion mice, and demonstrated that odontoblast β-catenin signaling regulates odontoblast proliferation and differentiation. However, the role of odontoblast β-catenin signaling in regulation of HERS behavior has not been fully investigated. Here, using the same odontoblast-specific Ctnnb1 deletion mice, we found that ablation of β-catenin signaling in odontoblasts led to aberrant HERS formation. Mechanistically, odontoblast-specific Ctnnb1 deletion resulted in elevated bone morphogenetic protein 7(Bmp7) expression and reduced expression of noggin and follistatin, both of which encode extracellular inhibitors of BMPs. Furthermore, the levels of phosphorylated Smad1/5/8 were increased in HERS cells. In vitro tissue culture confirmed that BMP7 treatment disrupted the HERS structure. Taken together, we demonstrated that odontoblast β-catenin signaling may act through regulation of BMP signaling to maintain the integrity of HERS cells.     

α-Actinin-4/FSGS1 is required for Arp2/3-dependent actin assembly at the adherens junction

We have developed an in vitro assay to study actin assembly at cadherin-enriched cell junctions. Using this assay, we demonstrate that cadherin-enriched junctions can polymerize new actin filaments but cannot capture preexisting filaments, suggesting a mechanism involving de novo synthesis. In agreement with this hypothesis, inhibition of Arp2/3-dependent nucleation abolished actin assembly at cell-cell junctions. Reconstitution biochemistry using the in vitro actin assembly assay identified α-actinin-4/focal segmental glomerulosclerosis 1 (FSGS1) as an essential factor. α-Actinin-4 specifically localized to sites of actin incorporation on purified membranes and at apical junctions in Madin-Darby canine kidney cells. Knockdown of α-actinin-4 decreased total junctional actin and inhibited actin assembly at the apical junction. Furthermore, a point mutation of α-actinin-4 (K255E) associated with FSGS failed to support actin assembly and acted as a dominant negative to disrupt actin dynamics at junctional complexes. These findings demonstrate that α-actinin-4 plays an important role in coupling actin nucleation to assembly at cadherin-based cell-cell adhesive contacts.     

The clathrin assembly protein AP180 regulates the generation of amyloid-β peptide

The overproduction and extracellular buildup of amyloid-β peptide (Aβ) is a critical step in the etiology of Alzheimer’s disease. Recent data suggest that intracellular trafficking is of central importance in the production of Aβ. Here we use a neuronal cell line to examine two structurally similar clathrin assembly proteins, AP180 and CALM. We show that RNA interference-mediated knockdown of AP180 reduces the generation of Aβ1-40 and Aβ1-42, whereas CALM knockdown has no effect on Aβ generation. Thus AP180 is among the traffic controllers that oversee and regulate amyloid precursor protein processing pathways. Our results also suggest that AP180 and CALM, while similar in their domain structures and biochemical properties, are in fact dedicated to separate trafficking pathways in neurons.     

Nonredundant roles of cytoplasmic β- and γ-actin isoforms in regulation of epithelial apical junctions

Association with the actin cytoskeleton is critical for normal architecture and dynamics of epithelial tight junctions (TJs) and adherens junctions (AJs). Epithelial cells express β-cytoplasmic (β-CYA) and γ-cytoplasmic (γ-CYA) actins, which have different cellular localization and functions. This study elucidates the roles of cytoplasmic actins in regulating structure and remodeling of AJs and TJs in model intestinal epithelia. Immunofluorescence labeling and latrunculin B treatment reveal affiliation of dynamic β-CYA filaments with newly assembled and mature AJs, whereas an apical γ-CYA pool is composed of stable perijunctional bundles and rapidly turning-over nonjunctional filaments. The functional effects of cytoplasmic actins on epithelial junctions are examined by using isoform-specific small interfering RNAs and cell-permeable inhibitory peptides. These experiments demonstrate unique roles of β-CYA and γ-CYA in regulating the steady-state integrity of AJs and TJs, respectively. Furthermore, β-CYA is selectively involved in establishment of apicobasal cell polarity. Both actin isoforms are essential for normal barrier function of epithelial monolayers, rapid AJ/TJ reassembly, and formation of three-dimensional cysts. Cytoplasmic actin isoforms play unique roles in regulating structure and permeability of epithelial junctions.     

The COP9 signalosome: an assembly and maintenance platform for cullin ubiquitin ligases?

The COP9 signalosome (CSN) is a highly conserved protein complex implicated in diverse biological functions that involve ubiquitin-mediated proteolysis. Paradoxically, conserved enzymatic activities associated with CSN inhibit cullin ubiquitin ligase activity in vitro, whereas mutational analysis suggests that CSN promotes cullin-dependent proteolysis in vivo. This apparent paradox can be resolved in a model that proposes CSN-mediated cullin inhibition is a prerequisite for the proper assembly and maintenance of active cullin ubiquitin ligase complexes.     

Laminin induced local axonal translation of β-actin mRNA is impaired in SMN-deficient motoneurons

Reduced levels of the SMN (survival of motoneuron) protein cause spinal muscular atrophy, the main form of motoneuron disease in children and young adults. In cultured motoneurons, reduced SMN levels lead to disturbed axon growth that correlates with reduced actin mRNA and protein in growth cones, indicating that anterograde transport and local translation of β-actin mRNA are altered in this disease. However, it is not fully understood how local translation of the β-actin mRNA is regulated in SMN-deficient motoneurons. Here, we established a lentiviral GFP-based reporter construct to monitor local translation of β-actin mRNA. Time-lapse imaging of fluorescence recovery after photobleaching (FRAP) in living motoneurons revealed that β-actin is locally translated in the growth cones of embryonic motoneurons. Interestingly, local translation of the β-actin reporter construct was differentially regulated by various Laminin isoforms, indicating that Laminins provide extracellular cues for the regulation of local translation in growth cones. Notably, local translation of β-actin mRNA was deregulated in motoneurons from a mouse model for the most severe form of SMA (Smn ^?/? ;SMN2). Taken together our findings suggest that local translation of β-actin in growth cones of motoneurons is regulated by Laminin signalling and that this signalling is disturbed in SMA.     

The β(3) -integrin ligand of Borrelia burgdorferi is critical for infection of mice but not ticks

P66 is a Borrelia burgdorferi surface protein with β₃ integrin binding and channel forming activities. In this study, the role of P66 in mammalian and tick infection was examined. B. burgdorferiΔp66 strains were not infectious in wild‐type, TLR2^?/?‐ or MyD88^?/?‐deficient mice. Strains with p66 restored to the chromosome restored near wild‐type infectivity, while complementation with p66 on a shuttle vector did not restore infectivity. Δp66 mutants are cleared quickly from the site of inoculation, but analyses of cytokine expression and cellular infiltrates at the site of inoculation did not reveal a specific mechanism of clearance. The defect in these mutants cannot be attributed to nutrient limitation or an inability to adapt to the host environment in vivo as Δp66 bacteria were able to survive as well as wild type in dialysis membrane chambers in the rat peritoneum. Δp66 bacteria were able to survive in ticks through the larva to nymph moult, but were non‐infectious in mice when delivered by tick bite. Independent lines of evidence do not support any increased susceptibility of the Δp66 strains to factors in mammalian blood. This study is the first to define a B. burgdorferi adhesin as essential for mammalian, but not tick infection.     

RACK1 is a ribosome scaffold protein for β-actin mRNA/ZBP1 complex

In neurons, specific mRNAs are transported in a translationally repressed manner along dendrites or axons by transport ribonucleic-protein complexes called RNA granules. ZBP1 is one RNA binding protein present in transport RNPs, where it transports and represses the translation of cotransported mRNAs, including β-actin mRNA. The release of β-actin mRNA from ZBP1 and its subsequent translation depends on the phosphorylation of ZBP1 by Src kinase, but little is known about how this process is regulated. Here we demonstrate that the ribosomal-associated protein RACK1, another substrate of Src, binds the β-actin mRNA/ZBP1 complex on ribosomes and contributes to the release of β-actin mRNA from ZBP1 and to its translation. We identify the Src binding and phosphorylation site Y246 on RACK1 as the critical site for the binding to the β-actin mRNA/ZBP1 complex. Based on these results we propose RACK1 as a ribosomal scaffold protein for specific mRNA-RBP complexes to tightly regulate the translation of specific mRNAs.     

Out but Not In: The Large Transmembrane β-Barrel Protein FhuA Unfolds but Cannot Refold via β-Hairpins

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Highlights? Mechanical un- and refolding studies of single large transmembrane β-barrel proteins ? FhuA unfolds via highly reproducible steps formed by single β-hairpins ? Once unfolded, FhuA folds into highly irreproducible structures ? To assist folding into the membrane, FhuA may require cofactors