Milk fat globule-epidermal growth factor 8 (MFGE8) prevents intestinal fibrosis.

OBJECTIVE: Intestinal fibrosis is considered an inevitable consequence of chronic IBD, leading to stricture formation and need for surgery. During the process of fibrogenesis, extracellular matrix (ECM) components critically regulate the function of mesenchymal cells. We characterised the composition and function of ECM in fibrostenosing Crohn's disease (CD) and control tissues. DESIGN: Decellularised full-thickness intestinal tissue platforms were tested using three different protocols, and ECM composition in different tissue phenotypes was explored by proteomics and validated by quantitative PCR (qPCR) and immunohistochemistry. Primary human intestinal myofibroblasts (HIMFs) treated with milk fat globule-epidermal growth factor 8 (MFGE8) were evaluated regarding the mechanism of their antifibrotic response, and the action of MFGE8 was tested in two experimental intestinal fibrosis models. RESULTS: We established and validated an optimal decellularisation protocol for intestinal IBD tissues. Matrisome analysis revealed elevated MFGE8 expression in CD strictured (CDs) tissue, which was confirmed at the mRNA and protein levels. Treatment with MFGE8 inhibited ECM production in normal control HIMF but not CDs HIMF. Next-generation sequencing uncovered functionally relevant integrin-mediated signalling pathways, and blockade of integrin αvß5 and focal adhesion kinase rendered HIMF non-responsive to MFGE8. MFGE8 prevented and reversed experimental intestinal fibrosis in vitro and in vivo. CONCLUSION: MFGE8 displays antifibrotic effects, and its administration may represent a future approach for prevention of IBD-induced intestinal strictures.

Genome-wide screens identify SEL1L as an intracellular rheostat controlling collagen turnover.

Accumulating evidence has implicated impaired extracellular matrix (ECM) clearance as a key factor in fibrotic disease. Despite decades of research elucidating the effectors of ECM clearance, relatively little is understood regarding the upstream regulation of this process. Collagen is the most abundant constituent of normal and fibrotic ECM in mammalian tissues. Its catabolism occurs through extracellular proteolysis and cell-mediated uptake of collagen fragments for intracellular degradation. Given the paucity of information regarding the regulation of this latter process, here we execute unbiased genome-wide screens to understand the molecular underpinnings of cell-mediated collagen clearance. Using this approach, we discover a mechanism through which collagen biosynthesis is sensed by cells internally and directly regulates clearance of extracellular collagen. The sensing mechanism appears to be dependent on endoplasmic reticulum-resident protein SEL1L and occurs via a noncanonical function of this protein. This pathway functions as a homeostatic negative feedback loop that limits collagen accumulation in tissues. In human fibrotic lung disease, the induction of this collagen clearance pathway by collagen synthesis is impaired, thereby contributing to the pathological accumulation of collagen in lung tissue. Thus, we describe cell-autonomous, rheostatic collagen clearance as an important pathway of tissue homeostasis.

PTP1B mediates the inhibitory effect of MFGE8 on insulin signaling through the ß5 integrin.

Integrins are cell adhesion receptors that dimerize to mediate cell-cell interactions and regulate processes, including proliferation, inflammation, and tissue repair. The role of integrins in regulating insulin signaling is incompletely understood. We have previously shown that binding of the integrin ligand milk fat globule epidermal growth factor like 8 (MFGE8) to the αvß5 integrin promotes termination of insulin receptor signaling in mice. Upon ligation of MFGE8, integrin ß5 complexes with the insulin receptor beta (IRß) in skeletal muscle, resulting in dephosphorylation of IRß and reduction of insulin-stimulated glucose uptake. Here, we investigate the mechanism by which the interaction between ß5 and IRß impacts IRß phosphorylation status. We show in in vitro and in vivo in skeletal muscle in mice that antibody-mediated blockade of the ß5 integrin inhibits and recombinant MFGE8 promotes PTP1B binding to and dephosphorylation of IRß resulting in increased or reduced insulin-stimulated glucose uptake, respectively. The ß5-PTP1B complex is recruited by MFGE8 to IRß leading to termination of canonical insulin signaling. ß5 blockade enhances insulin-stimulated glucose uptake in wildtype but not Ptp1b KO mice indicating that PTP1B functions downstream of MFGE8 in modulating insulin receptor signaling. Furthermore, in a human cohort, we report serum MFGE8 levels correlate with indices of insulin resistance. These data provide mechanistic insights into the role of MFGE8 and ß5 in regulating insulin signaling.

BCL-2 Modulates IRE1α Activation to Attenuate Endoplasmic Reticulum Stress and Pulmonary Fibrosis.

BCL-2 family members are known to be implicated in survival in numerous biological settings. Here, we provide evidence that in injury and repair processes in lungs, BCL-2 mainly acts to attenuate endoplasmic reticulum (ER) stress and limit extracellular matrix accumulation. Days after an intratracheal bleomycin challenge, mice lose a fraction of their alveolar type II epithelium from terminal ER stress driven by activation of the critical ER sensor and stress effector IRE1α. This fraction is dramatically increased by BCL-2 inhibition, because IRE1α activation is dependent on its physical association with the BCL-2-proapoptotic family member BAX, and we found BCL-2 to disrupt this association in vitro. In vivo, navitoclax (a BCL-2/BCL-xL inhibitor) given 15-21 days after bleomycin challenge evoked strong activation of IRE-1α in mesenchymal cells and markers of ER stress, but not apoptosis. Remarkably, after BCL-2 inhibition, bleomycin-exposed mice demonstrated persistent collagen accumulation at Day 42, compared with resolution in controls. Enhanced fibrosis proved to be due to the RNAase activity of IRE1α downregulating MRC2 mRNA and protein, a mediator of collagen turnover. The critical role of MRC2 was confirmed in precision-cut lung slice cultures of Day-42 lungs from bleomycin-exposed wild-type and MRC2 null mice. Soluble and tissue collagen accumulated in precision-cut lung slice cultures from navitoclax-treated, bleomycin-challenged mice compared with controls, in a manner nearly identical to that of challenged but untreated MRC2 null mice. Thus, apart from mitochondrial-based antiapoptosis, BCL-2 functions to attenuate ER stress responses, fostering tissue homeostasis and injury repair.

Radial glia promote microglial development through integrin αVß8 -TGFß1 signaling.

Microglia diversity emerges from interactions between intrinsic genetic programs and environment-derived signals, but how these processes unfold and interact in the developing brain remains unclear. Here, we show that radial glia-expressed integrin beta 8 (ITGB8) expressed in radial glia progenitors activates microglia-expressed TGFß1, permitting microglial development. Domain-restricted deletion of Itgb8 in these progenitors establishes complementary regions with developmentally arrested "dysmature" microglia that persist into adulthood. In the absence of autocrine TGFß1 signaling, we find that microglia adopt a similar dysmature phenotype, leading to neuromotor symptoms almost identical to Itgb8 mutant mice. In contrast, microglia lacking the TGFß signal transducers Smad2 and Smad3 have a less polarized dysmature phenotype and correspondingly less severe neuromotor dysfunction. Finally, we show that non-canonical (Smad-independent) signaling partially suppresses disease and development associated gene expression, providing compelling evidence for the adoption of microglial developmental signaling pathways in the context of injury or disease.

MFGE8 inhibits insulin signaling through PTP1B.

The role of integrins in regulating insulin signaling is incompletely understood. We have previously shown that binding of the integrin ligand milk fat globule epidermal growth factor like 8 (MFGE8) to the αvß5 integrin promotes termination of insulin receptor signaling in mice. Upon ligation of MFGE8, ß5 complexes with the insulin receptor beta (IRß) in skeletal muscle resulting in dephosphorylation of IRß and reduction of insulin-stimulated glucose uptake. Here we investigate the mechanism by which the interaction between ß5 and IRß impacts IRß phosphorylation status. We show that ß5 blockade inhibits and MFGE8 promotes PTP1B binding to and dephosphorylation of IRß resulting in reduced or increased insulin-stimulated myotube glucose uptake respectively. The ß5-PTP1B complex is recruited by MFGE8 to IRß leading to termination of canonical insulin signaling. ß5 blockade enhances insulin-stimulated glucose uptake in wild type but not Ptp1b KO mice indicating that PTP1B functions downstream of MFGE8 in modulating insulin receptor signaling. Furthermore, in a human cohort, we report serum MFGE8 levels correlate with indices of insulin resistance. These data provide mechanistic insights into the role of MFGE8 and ß5 in regulating insulin signaling.

MFGE8 links absorption of dietary fatty acids with catabolism of enterocyte lipid stores through HNF4γ-dependent transcription of CES enzymes.

Enterocytes modulate the extent of postprandial lipemia by storing dietary fats in cytoplasmic lipid droplets (cLDs). We have previously shown that the integrin ligand MFGE8 links absorption of dietary fats with activation of triglyceride (TG) hydrolases that catabolize cLDs for chylomicron production. Here, we identify CES1D as the key hydrolase downstream of the MFGE8-αvß5 integrin pathway that regulates catabolism of diet-derived cLDs. Mfge8 knockout (KO) enterocytes have reduced CES1D transcript and protein levels and reduced protein levels of the transcription factor HNF4γ. Both Ces1d and Hnf4γ KO mice have decreased enterocyte TG hydrolase activity coupled with retention of TG in cLDs. Mechanistically, MFGE8-dependent fatty acid uptake through CD36 stabilizes HNF4γ protein level; HNF4γ then increases Ces1d transcription. Our work identifies a regulatory network that regulates the severity of postprandial lipemia by linking dietary fat absorption with protein stabilization of a transcription factor that increases expression of hydrolases responsible for catabolizing diet-derived cLDs.

Genome-wide screens identify SEL1L as an intracellular rheostat controlling collagen turnover.

Accumulating evidence has implicated impaired extracellular matrix (ECM) clearance as a key factor in fibrotic disease. Despite decades of research elucidating the effectors of ECM clearance, relatively little is understood regarding the upstream regulation of this process. Collagen is the most abundant constituent of normal and fibrotic ECM in mammalian tissues. Its catabolism occurs through extracellular proteolysis and cell-mediated uptake of collagen fragments for intracellular degradation. Given the paucity of information regarding the regulation of this latter process, we executed unbiased genome-wide screens to understand the molecular underpinnings of cell-mediated collagen clearance. Using this approach, we discovered a previously unappreciated mechanism through which collagen biosynthesis is sensed by cells internally and directly regulates clearance of extracellular collagen. The sensing mechanism is dependent on endoplasmic reticulum-resident protein SEL1L and occurs via a noncanonical function of SEL1L. This pathway functions as a homeostatic negative feedback loop that limits collagen accumulation in tissues. In human fibrotic lung disease, the induction of this collagen clearance pathway by collagen synthesis is impaired, thereby contributing to the pathological accumulation of collagen in lung tissue. Thus cell-autonomous, rheostatic collagen clearance is a previously unidentified pathway of tissue homeostasis.

Autoregulation of insulin receptor signaling through MFGE8 and the αvß5 integrin.

The role of integrins, in particular αv integrins, in regulating insulin resistance is incompletely understood. We have previously shown that the αvß5 integrin ligand milk fat globule epidermal growth factor like 8 (MFGE8) regulates cellular uptake of fatty acids. In this work, we evaluated the impact of MFGE8 on glucose homeostasis. We show that acute blockade of the MFGE8/ß5 pathway enhances while acute augmentation dampens insulin-stimulated glucose uptake. Moreover, we find that insulin itself induces cell-surface enrichment of MFGE8 in skeletal muscle, which then promotes interaction between the αvß5 integrin and the insulin receptor leading to dampening of skeletal-muscle insulin receptor signaling. Blockade of the MFGE8/ß5 pathway also enhances hepatic insulin sensitivity. Our work identifies an autoregulatory mechanism by which insulin-stimulated signaling through its cognate receptor is terminated through up-regulation of MFGE8 and its consequent interaction with the αvß5 integrin, thereby establishing a pathway that can potentially be targeted to improve insulin sensitivity.

You Say You Want a Resolution (of Fibrosis).

In pathological fibrosis, aberrant tissue remodeling with excess extracellular matrix leads to organ dysfunction and eventual morbidity. Diseases of fibrosis create significant global health and economic burdens and are often deadly. Although fibrosis has traditionally been thought of as an irreversible process, a growing body of evidence demonstrates that organ fibrosis can reverse in certain circumstances, especially if an underlying cause of injury can be removed. This body of evidence has uncovered more and more contributors to persistent and nonresolving tissue fibrosis. Here, we review the present knowledge on resolution of organ fibrosis and restoration of near-normal tissue architecture. We emphasize three critical areas of tissue homeostasis that are necessary for fibrosis resolution, namely, the elimination of matrix-producing cells, the clearance of excess matrix, and the regeneration of normal tissue constituents. In so doing, we also highlight how profibrotic pathways interact with one another and where there may be therapeutic opportunities to intervene and remediate pathological persistent fibrosis.

CD81 Controls Beige Fat Progenitor Cell Growth and Energy Balance via FAK Signaling.

Adipose tissues dynamically remodel their cellular composition in response to external cues by stimulating beige adipocyte biogenesis; however, the developmental origin and pathways regulating this process remain insufficiently understood owing to adipose tissue heterogeneity. Here, we employed single-cell RNA-seq and identified a unique subset of adipocyte progenitor cells (APCs) that possessed the cell-intrinsic plasticity to give rise to beige fat. This beige APC population is proliferative and marked by cell-surface proteins, including PDGFRα, Sca1, and CD81. Notably, CD81 is not only a beige APC marker but also required for de novo beige fat biogenesis following cold exposure. CD81 forms a complex with αV/ß1 and αV/ß5 integrins and mediates the activation of integrin-FAK signaling in response to irisin. Importantly, CD81 loss causes diet-induced obesity, insulin resistance, and adipose tissue inflammation. These results suggest that CD81 functions as a key sensor of external inputs and controls beige APC proliferation and whole-body energy homeostasis.

Age-dependent regulation of cell-mediated collagen turnover.

Although aging represents the most important epidemiologic risk factor for fibrotic disease, the reasons for this are incompletely understood. Excess collagen deposition in tissues is the sine qua non of tissue fibrosis and can be viewed as an imbalance between collagen production and collagen degradation. Yet we still lack a detailed understanding of the changes that take place during development, maturation, and aging in extracellular matrix (ECM) dynamics. Resolution of fibrosis is impaired in aging, and this impairment may explain why age is the most important risk factor for fibrotic diseases, such as idiopathic pulmonary fibrosis. However, ECM dynamics and impaired resolution of fibrosis in aging remain understudied. Here we show that cell-mediated collagen uptake and degradation are diminished in aged animals and this finding correlates with downregulation of the collagen endocytic receptor mannose receptor, C-type 2 (Mrc2). We identify myeloid zinc finger-1 as a potentially novel transcriptional regulator of Mrc2, and both this transcription factor and Mrc2 are downregulated in multiple tissues and organisms in an age-dependent manner. Thus, cell-mediated degradation of collagen is an essential process that promotes resolution of fibrosis, and impairment in this process contributes to age-related fibrosis.

Fat fibrosis: friend or foe?

At the simplest level, obesity is the manifestation of an imbalance between caloric intake and expenditure; however, the pathophysiological mechanisms that govern the development of obesity and associated complications are enormously complex. Fibrosis within the adipose tissue compartment is one such factor that may influence the development of obesity and/or obesity-related comorbidities. Furthermore, the functional consequences of adipose tissue fibrosis are a matter of considerable debate, with evidence that fibrosis serves both adaptive and maladaptive roles. Tissue fibrosis itself is incompletely understood, and multiple cellular and molecular pathways are involved in the development, maintenance, and resolution of the fibrotic state. Within the context of obesity, fibrosis influences molecular and cellular events that relate to adipocytes, inflammatory cells, inflammatory mediators, and supporting adipose stromal tissue. In this Review, we explore what is known about the interplay between the development of adipose tissue fibrosis and obesity, with a view toward future investigative and therapeutic avenues.

Autocrine Mfge8 Signaling Prevents Developmental Exhaustion of the Adult Neural Stem Cell Pool.

Adult neurogenesis, arising from quiescent radial-glia-like neural stem cells (RGLs), occurs throughout life in the dentate gyrus. How neural stem cells are maintained throughout development to sustain adult mammalian neurogenesis is not well understood. Here, we show that milk fat globule-epidermal growth factor (EGF) 8 (Mfge8), a known phagocytosis factor, is highly enriched in quiescent RGLs in the dentate gyrus. Mfge8-null mice exhibit decreased adult dentate neurogenesis, and furthermore, adult RGL-specific deletion of Mfge8 leads to RGL overactivation and depletion. Similarly, loss of Mfge8 promotes RGL activation in the early postnatal dentate gyrus, resulting in a decreased number of label-retaining RGLs in adulthood. Mechanistically, loss of Mfge8 elevates mTOR1 signaling in RGLs, inhibition of which by rapamycin returns RGLs to quiescence. Together, our study identifies a neural-stem-cell-enriched niche factor that maintains quiescence and prevents developmental exhaustion of neural stem cells to sustain continuous neurogenesis in the adult mammalian brain.

Cell division cycle 7 kinase is a negative regulator of cell-mediated collagen degradation.

Although extensive work has delineated many of the mechanisms of extracellular matrix (ECM) production, far less is known about pathways that regulate ECM degradation. This is particularly true of cellular internalization and degradation of matrix, which play an underappreciated role in ECM metabolism and lung fibrosis. For example, genetic perturbation of this pathway leads to exacerbated fibrosis in experimental animal models. In this work, we present the results of an unbiased screen of Drosophila phagocytes that yielded multiple genes that, when silenced, led to increased collagen uptake. We further describe the function of cell division cycle 7 kinase (CDC7) as a specific suppressor of collagen uptake. We show that the genetic or pharmacological inhibition of CDC7 results in increased expression of the collagen endocytic receptor Endo180. Chromobox 5 (CBX5) is a putative target of CDC7, and genetic silencing of CBX5 also results in increased Endo180 and collagen uptake. Finally, CRISPR-mediated activation of Endo180 expression results in increased collagen uptake, suggesting that CDC7 regulates collagen internalization through increased Endo180 expression. Targeting the regulatory elements of the collagen degradative machinery may be a useful therapeutic approach in diseases of fibrosis or malignancy.

The Mfge8-α8ß1-PTEN pathway regulates airway smooth muscle contraction in allergic inflammation.

Asthma affects ∼300 million people worldwide. Despite multiple treatment options, asthma treatment remains unsatisfactory in a subset of patients. Airway obstruction is a hallmark of allergic asthma and is largely due to airway smooth muscle hypercontractility induced by airway inflammation. Identification of molecular pathways that regulate airway smooth muscle hypercontractility is of considerable therapeutic interest. We previously identified roles for milk fat globule epidermal growth factor-like 8 (Mfge8) in opposing the effects of allergic inflammation on increasing airway smooth muscle contractile force. In this study, we delineate the signaling pathway by which Mfge8 mediates these effects. By using genetic and pharmacologic approaches, we show that the α8ß1 integrin and the phosphatase and tensin homolog (PTEN) mediate the effects of Mfge8 on preventing IL-13-induced increases in airway contractility. Tracheal rings from mice with smooth muscle-specific deletion of α8ß1 or PTEN have enhanced contraction in response to treatment with IL-13. Enhanced IL-13-induced tracheal ring contraction in Mfge8-/- mice was abolished by treatment with the PI3K inhibitor. Mechanistically, IL-13 induces ubiquitination and degradation of PTEN protein. Our findings identify a role for the Mfge8-α8ß1-PTEN pathway in regulating the force of airway smooth muscle contraction in the setting of allergic inflammation.-Khalifeh-Soltani, A., Gupta, D., Ha, A., Podolsky, M. J., Datta, R., Atabai, K. The Mfge8-α8ß1-PTEN pathway regulates airway smooth muscle contraction in allergic inflammation.

Extracellular matrix in lung development, homeostasis and disease.

The lung's unique extracellular matrix (ECM), while providing structural support for cells, is critical in the regulation of developmental organogenesis, homeostasis and injury-repair responses. The ECM, via biochemical or biomechanical cues, regulates diverse cell functions, fate and phenotype. The composition and function of lung ECM become markedly deranged in pathological tissue remodeling. ECM-based therapeutics and bioengineering approaches represent promising novel strategies for regeneration/repair of the lung and treatment of chronic lung diseases. In this review, we assess the current state of lung ECM biology, including fundamental advances in ECM composition, dynamics, topography, and biomechanics; the role of the ECM in normal and aberrant lung development, adult lung diseases and autoimmunity; and ECM in the regulation of the stem cell niche. We identify opportunities to advance the field of lung ECM biology and provide a set recommendations for research priorities to advance knowledge that would inform novel approaches to the pathogenesis, diagnosis, and treatment of chronic lung diseases.

Inflammatory bone loss associated with MFG-E8 deficiency is rescued by teriparatide.

A prolonged increase in proinflammatory cytokines is associated with osteoporotic and autoimmune bone loss and, conversely, anti-inflammatory pathways are associated with protection against bone loss. Milk fat globule-epidermal growth factor (MFG-E)-8 is a glycoprotein that is proresolving, regulates apoptotic cell clearance, and has been linked to autoimmune disease and skeletal homeostasis. The role of MFG-E8 in the young vs. adult skeleton was determined in mice deficient in MFG-E8 (KO). In vivo, trabecular bone was similar in MFG-E8KO and wild-type (WT) mice at 6 and 16 wk, whereas 22 wk adult MFG-E8KO mice displayed significantly reduced trabecular BV/TV. The number of osteoclasts per bone surface was increased in 22-wk MFG-E8 KO vs. WT mice, and recombinant murine MFG-E8 decreased the number and size of osteoclasts in vitro. Adult MFG-E8KO spleen weight:body weight was increased compared with WT, and flow cytometric analysis showed significantly increased myeloid-derived suppressor cells (CD11bhiGR-1+) and neutrophils (CD11bhiLy6G+) in MFG-E8KO bone marrow, suggesting an inflammatory phenotype. PTH-treated MFG-E8KO mice showed a greater anabolic response (+124% BV/TV) than observed in PTH-treated WT mice (+64% BV/TV). These data give insight into the role of MFG-E8 in the adult skeleton and suggest that anabolic PTH may be a valuable therapeutic approach for autoimmune-associated skeletal disease.-Michalski, M. N., Seydel, A. L., Siismets, E. M., Zweifler, L. E., Koh, A. J., Sinder, B. P., Aguirre, J. I., Atabai, K., Roca, H., McCauley, L. K. Inflammatory bone loss associated with MFG-E8 deficiency is rescued by teriparatide.