R. Grant Rowe

Snail1-induced partial epithelial-to-mesenchymal transition drives renal fibrosis in mice and can be targeted to reverse established disease

Progressive kidney fibrosis contributes greatly to end-stage renal failure, and no specific treatment is available to preserve organ function. During renal fibrosis, myofibroblasts accumulate in the interstitium of the kidney, leading to massive deposition of extracellular matrix and organ dysfunction. The origin of myofibroblasts is manifold, but the contribution of an epithelial-to-mesenchymal transition (EMT) undergone by renal epithelial cells during kidney fibrosis is still debated. We show that the reactivation of Snai1 (encoding snail family zinc finger 1, known as Snail1) in mouse renal epithelial cells is required for the development of fibrosis in the kidney. Damage-mediated Snail1 reactivation induces a partial EMT in tubular epithelial cells that, without directly contributing to the myofibroblast population, relays signals to the interstitium to promote myofibroblast differentiation and fibrogenesis and to sustain inflammation. We also show that Snail1-induced fibrosis can be reversed in vivo and that obstructive nephropathy can be therapeutically ameliorated in mice by targeting Snail1 expression. These results reconcile conflicting data on the role of the EMT in renal fibrosis and provide avenues for the design of novel anti-fibrotic therapies.

Fibronectin fibrillogenesis regulates three-dimensional neovessel formation

During vasculogenesis and angiogenesis, endothelial cell responses to growth factors are modulated by the compositional and mechanical properties of a surrounding three-dimensional (3D) extracellular matrix (ECM) that is dominated by either cross-linked fibrin or type I collagen. While 3D-embedded endothelial cells establish adhesive interactions with surrounding ligands to optimally respond to soluble or matrix-bound agonists, the manner in which a randomly ordered ECM with diverse physico-mechanical properties is remodeled to support blood vessel formation has remained undefined. Herein, we demonstrate that endothelial cells initiate neovascularization by unfolding soluble fibronectin (Fn) and depositing a pericellular network of fibrils that serve to support cytoskeletal organization, actomyosin-dependent tension, and the viscoelastic properties of the embedded cells in a 3D-specific fashion. These results advance a new model wherein Fn polymerization serves as a structural scaffolding that displays adhesive ligands on a mechanically ideal substratum for promoting neovessel development.

Navigating ECM Barriers at the Invasive Front: The Cancer Cell–Stroma Interface

A seminal event in cancer progression is the ability of the neoplastic cell to mobilize the necessary machinery to breach surrounding extracellular matrix barriers while orchestrating a host stromal response that ultimately supports tissue-invasive and metastatic processes. With over 500 proteolytic enzymes identified in the human genome, interconnecting webs of protease-dependent and protease-independent processes have been postulated to drive the cancer cell invasion program via schemes of daunting complexity. Increasingly, however, a body of evidence has begun to emerge that supports a unifying model wherein a small group of membrane-tethered enzymes, termed the membrane-type matrix metalloproteinases (MT-MMPs), plays a dominant role in regulating cancer cell, as well as stromal cell, traffic through the extracellular matrix barriers assembled by host tissues in vivo. Understanding the mechanisms that underlie the regulation and function of these metalloenzymes as host cell populations traverse the dynamic extracellular matrix assembled during neoplastic states should provide new and testable theories regarding cancer invasion and metastasis.

Secreted Versus Membrane-anchored Collagenases RELATIVE ROLES IN FIBROBLAST-DEPENDENT COLLAGENOLYSIS AND INVASION

Fibroblasts degrade type I collagen, the major extracellular protein found in mammals, during events ranging from bulk tissue resorption to invasion through the three-dimensional extracellular matrix. Current evidence suggests that type I collagenolysis is mediated by secreted as well as membrane-anchored members of the matrix metalloproteinase (MMP) gene family. However, the roles played by these multiple and possibly redundant, degradative systems during fibroblast-mediated matrix remodeling is undefined. Herein, we use fibroblasts isolated from Mmp13−/−, Mmp8−/−, Mmp2−/−, Mmp9−/−, Mmp14−/− and Mmp16−/− mice to define the functional roles for secreted and membrane-anchored collagenases during collagen-resorptive versus collagen-invasive events. In the presence of a functional plasminogen activator-plasminogen axis, secreted collagenases arm cells with a redundant collagenolytic potential that allows fibroblasts harboring single deficiencies for either MMP-13, MMP-8, MMP-2, or MMP-9 to continue to degrade collagen comparably to wild-type fibroblasts. Likewise, Mmp14−/− or Mmp16−/− fibroblasts retain near-normal collagenolytic activity in the presence of plasminogen via the mobilization of secreted collagenases, but only Mmp14 (MT1-MMP) plays a required role in the collagenolytic processes that support fibroblast invasive activity. Furthermore, by artificially tethering a secreted collagenase to the surface of Mmp14−/− fibroblasts, we demonstrate that localized pericellular collagenolytic activity differentiates the collagen-invasive phenotype from bulk collagen degradation. Hence, whereas secreted collagenases arm fibroblasts with potent matrix-resorptive activity, only MT1-MMP confers the focal collagenolytic activity necessary for supporting the tissue-invasive phenotype.

Mesenchymal cells reactivate Snail1 expression to drive three-dimensional invasion programs

Epithelial–mesenchymal transition (EMT) is required for mesodermal differentiation during development. The zinc-finger transcription factor, Snail1, can trigger EMT and is sufficient to transcriptionally reprogram epithelial cells toward a mesenchymal phenotype during neoplasia and fibrosis. Whether Snail1 also regulates the behavior of terminally differentiated mesenchymal cells remains unexplored. Using a Snai1 conditional knockout model, we now identify Snail1 as a regulator of normal mesenchymal cell function. Snail1 expression in normal fibroblasts can be induced by agonists known to promote proliferation and invasion in vivo. When challenged within a tissue-like, three-dimensional extracellular matrix, Snail1-deficient fibroblasts exhibit global alterations in gene expression, which include defects in membrane type-1 matrix metalloproteinase (MT1-MMP)-dependent invasive activity. Snail1-deficient fibroblasts explanted atop the live chick chorioallantoic membrane lack tissue-invasive potential and fail to induce angiogenesis. These findings establish key functions for the EMT regulator Snail1 after terminal differentiation of mesenchymal cells.

MT1-MMP-Dependent Control of Skeletal Stem Cell Commitment via a β1-Integrin/YAP/TAZ Signaling Axis

In vitro, topographical and biophysical cues arising from the extracellular matrix (ECM) direct skeletal stem cell (SSC) commitment and differentiation. However, the mechanisms by which the SSC-ECM interface is regulated and the outcome of such interactions on stem cell fate in vivo remain unknown. Here we demonstrate that conditional deletion of the membrane-anchored metalloproteinase MT1-MMP (Mmp14) in mesenchymal progenitors, but not in committed osteoblasts, redirects SSC fate decisions from osteogenesis to adipo- and chondrogenesis. By effecting ECM remodeling, MT1-MMP regulates stem cell shape, thereby activating a β1-integrin/RhoGTPase signaling cascade and triggering the nuclear localization of the transcriptional coactivators YAP and TAZ, which serve to control SSC lineage commitment. These data identify a critical MT1-MMP/integrin/YAP/TAZ axis operative in the stem cell niche that oversees SSC fate determination.

Hepatocyte-derived snail1 propagates liver fibrosis progression

ABSTRACT Chronic exposure of the liver to hepatotoxic agents initiates an aberrant wound healing response marked by proinflammatory, as well as fibrotic, changes, leading to compromised organ structure and function. In a variety of pathological states, correlative links have been established between tissue fibrosis and the expression of transcription factors associated with the induction of epithelial-mesenchymal cell transition (EMT) programs similar to those engaged during development. However, the role played by endogenously derived, EMT-associated transcription factors in fibrotic states in vivo remains undefined. Using a mouse model of acute liver fibrosis, we demonstrate that hepatocytes upregulate the expression of the zinc finger transcriptional repressor, Snail1, during tissue remodeling. Hepatocyte-specific ablation of Snail1 demonstrates that this transcription factor plays a key role in liver fibrosis progression in vivo by triggering the proximal genetic programs that control multiple aspects of fibrogenesis, ranging from growth factor expression and extracellular matrix biosynthesis to the ensuing chronic inflammatory responses that characterize this class of pathological disorders.

Haematopoietic stem and progenitor cells from human pluripotent stem cells

A variety of tissue lineages can be differentiated from pluripotent stem cells by mimicking embryonic development through stepwise exposure to morphogens, or by conversion of one differentiated cell type into another by enforced expression of master transcription factors. Here, to yield functional human haematopoietic stem cells, we perform morphogen-directed differentiation of human pluripotent stem cells into haemogenic endothelium followed by screening of 26 candidate haematopoietic stem-cell-specifying transcription factors for their capacity to promote multi-lineage haematopoietic engraftment in mouse hosts. We recover seven transcription factors (ERG, HOXA5, HOXA9, HOXA10, LCOR, RUNX1 and SPI1) that are sufficient to convert haemogenic endothelium into haematopoietic stem and progenitor cells that engraft myeloid, B and T cells in primary and secondary mouse recipients. Our combined approach of morphogen-driven differentiation and transcription-factor-mediated cell fate conversion produces haematopoietic stem and progenitor cells from pluripotent stem cells and holds promise for modelling haematopoietic disease in humanized mice and for therapeutic strategies in genetic blood disorders.

MT1-MMP controls human mesenchymal stem cell trafficking and differentiation

Human mesenchymal stem cells (hMSCs) localized to bone marrow, nonhematopoietic organs, as well as perivascular niches are postulated to traffic through type I collagen-rich stromal tissues to first infiltrate sites of tissue damage, inflammation, or neoplasia and then differentiate. Nevertheless, the molecular mechanisms supporting the ability of hMSCs to remodel 3-dimensional (3D) collagenous barriers during trafficking or differentiation remain undefined. Herein, we demonstrate that hMSCs degrade and penetrate type I collagen networks in tandem with the expression of a 5-member set of collagenolytic matrix metalloproteinases (MMPs). Specific silencing of each of these proteases reveals that only a single membrane-tethered metalloenzyme, termed MT1-MMP, plays a required role in hMSC-mediated collagenolysis, 3D invasion, and intravasation. Further, once confined within type I collagen-rich tissue, MT1-MMP also controls hMSC differentiation in a 3D-specific fashion. Together, these data demonstrate that hMSC invasion and differentiation programs fall under the control of the pericellular collagenase, MT1-MMP.

Interactions between the secreted protein Amalgam, its transmembrane receptor Neurotactin and the Abelson tyrosine kinase affect axon pathfinding.

Two novel dosage-sensitive modifiers of the Abelson tyrosine kinase (Abl) mutant phenotype have been identified. Amalgam (Ama) is a secreted protein that interacts with the transmembrane protein Neurotactin (Nrt) to promote cell:cell adhesion. We have identified an unusual missense ama allele, amaM109, which dominantly enhances the Abl mutant phenotype, affecting axon pathfinding. Heterozygous null alleles of ama do not show this dominant enhancement, but animals homozygous mutant for both ama and Abl show abnormal axon outgrowth. Cell culture experiments demonstrate the AmaM109 mutant protein binds to Nrt, but is defective in mediating Ama/Nrt cell adhesion. Heterozygous null alleles of nrt dominantly enhance the Abl mutant phenotype, also affecting axon pathfinding. Furthermore, we have found that all five mutations originally attributed to disabled are in fact alleles of nrt. These results suggest Ama/Nrt-mediated adhesion may be part of signaling networks involving the Abl tyrosine kinase in the growth cone.