Hypusinated eIF5A is required for the translation of collagen.

Translation of mRNAs that encode peptide sequences with consecutive prolines (polyproline) requires the conserved and essential elongation factor eIF5A to facilitate the formation of peptide bonds. It has been shown that, upon eIF5A depletion, yeast ribosomes stall in polyproline motifs, but also in tripeptide sequences that combine proline with glycine and charged amino acids. Mammalian collagens are enriched in putative eIF5A-dependent Pro-Gly-containing tripeptides. Here, we show that depletion of active eIF5A in mouse fibroblasts reduced collagen type I α1 chain (Col1a1) content, which concentrated around the nuclei. Moreover, it provoked the upregulation of endoplasmic reticulum (ER) stress markers, suggesting retention of partially synthesized collagen 1 (Col1) in the ER. We confirmed that eIF5A is needed for heterologous collagen synthesis in yeast and, using a double luciferase reporter system, showed that eIF5A depletion interrupts translation at Pro-Gly collagenic motifs. A dramatically lower level of Col1a1 protein was also observed in functional eIF5A-depleted human hepatic stellate cells treated with the profibrotic cytokine TGF-ß1. In sum, our results show that collagen expression requires eIF5A and imply its potential as a target for regulating collagen production in fibrotic diseases.

Profilin1 is expressed in osteocytes and regulates cell shape and migration.

Osteocytes are the most abundant cells in bone and regulate bone metabolism in coordination with osteoblasts and osteoclasts. However, the molecules that control osteocytes are still incompletely understood. Profilin1 is an actin-binding protein that is involved in actin polymerization. Osteocytes possess characteristic dendritic process formed based on actin cytoskeleton. Here, we examined the expression of profilin1 and its function in osteocytes. Profilin1 mRNA was expressed in osteocytic MLO-Y4 cells and its levels were gradually increased along with the time in culture. With regard to functional aspect, knockdown of profilin1 by siRNA enhanced BMP-induced increase in alkaline phosphatase expression levels in MLO-Y4 cells. Profilin1 knockdown suppressed the levels of dendritic processes and migration of MLO-Y4 cells. Since aging causes an increase in ROS in the body, we further examined the effects of hydrogen peroxide on the expression of profilin1. Hydrogen peroxide treatment increased the levels of profilin1 mRNA in MLO-Y4 cells in contrast to the decline in alkaline phosphatase. Profilin1 was expressed not only in MLO-Y4cells but also in the primary cultures of osteocytes. Importantly, profilin1 mRNA levels in primary cultures of osteocytes were higher than those in primary cultures of osteoblasts. To examine in vivo role of profilin1 in osteocytes, profilin1 was conditionally knocked out by using DMP1-cre and profilin1 floxed mice. This conditional deletion of profilin1 specifically in osteocytes resulted in reduction in the levels of bone volume and bone mineral density. These data indicate that profilin1 is expressed in osteocytes and regulates cell shape, migration and bone mass.

Fibronectin-bound α5ß1 integrins sense load and signal to reinforce adhesion in less than a second.

Integrin-mediated mechanosensing of the extracellular environment allows cells to control adhesion and signalling. Whether cells sense and respond to force immediately upon ligand-binding is unknown. Here, we report that during adhesion initiation, fibroblasts respond to mechanical load by strengthening integrin-mediated adhesion to fibronectin (FN) in a biphasic manner. In the first phase, which depends on talin and kindlin as well as on the actin nucleators Arp2/3 and mDia, FN-engaged α5ß1 integrins activate focal adhesion kinase (FAK) and c-Src in less than 0.5 s to steeply strengthen α5ß1- and αV-class integrin-mediated adhesion. When the mechanical load exceeds a certain threshold, fibroblasts decrease adhesion and initiate the second phase, which is characterized by less steep adhesion strengthening. This unique, biphasic cellular adhesion response is mediated by α5ß1 integrins, which form catch bonds with FN and signal to FN-binding integrins to reinforce cell adhesion much before visible adhesion clusters are formed.

Corrigendum: Fibronectin-bound α5ß1 integrins sense load and signal to reinforce adhesion in less than a second.

This corrects the article DOI: 10.1038/nmat5023.

Profilin 1 is required for peripheral nervous system myelination.

Myelination allows rapid saltatory propagation of action potentials along the axon and is an essential prerequisite for the normal functioning of the nervous system. During peripheral nervous system (PNS) development, myelin-forming Schwann cells (SCs) generate radial lamellipodia to sort and ensheath axons. This process requires controlled cytoskeletal remodeling, and we show that SC lamellipodia formation depends on the function of profilin 1 (Pfn1), an actin-binding protein involved in microfilament polymerization. Pfn1 is inhibited upon phosphorylation by ROCK, a downstream effector of the integrin linked kinase pathway. Thus, a dramatic reduction of radial lamellipodia formation is observed in SCs lacking integrin-linked kinase or treated with the Rho/ROCK activator lysophosphatidic acid. Knocking down Pfn1 expression by lentiviral-mediated shRNA delivery impairs SC lamellipodia formation in vitro, suggesting a direct role for this protein in PNS myelination. Indeed, SC-specific gene ablation of Pfn1 in mice led to profound radial sorting and myelination defects, confirming a central role for this protein in PNS development. Our data identify Pfn1 as a key effector of the integrin linked kinase/Rho/ROCK pathway. This pathway, acting in parallel with integrin ß1/LCK/Rac1 and their effectors critically regulates SC lamellipodia formation, radial sorting and myelination during peripheral nervous system maturation.

Profilin 1 is essential for retention and metabolism of mouse hematopoietic stem cells in bone marrow.

How stem cells interact with the microenvironment to regulate their cell fates and metabolism is largely unknown. Here we demonstrated that the deletion of the cytoskeleton-modulating protein profilin 1 (pfn1) in hematopoietic stem cell (HSCs) led to bone marrow failure, loss of quiescence, and mobilization and apoptosis of HSCs in vivo. A switch from glycolysis to mitochondrial respiration with increased reactive oxygen species (ROS) level was also observed in HSCs on pfn1 deletion. Importantly, treatment of pfn1-deficient mice with the antioxidant N-acetyl-l-cysteine reversed the ROS level and loss of quiescence of HSCs, suggesting that the metabolism is mechanistically linked to the cell cycle quiescence of stem cells. The actin-binding and proline-binding activities of pfn1 are required for its function in HSCs. Our study provided evidence that pfn1 at least partially acts through the axis of pfn1/Gα13/EGR1 to regulate stem cell retention and metabolism in the bone marrow.

The role of integrin binding sites in fibronectin matrix assembly in vivo.

The extracellular matrix (ECM) glycoprotein fibronectin (FN) requires the help of cells to assemble into a functional fibrillar matrix, which then orchestrates the assembly of other ECM proteins and promotes cell adhesion, migration and signalling. Fibrillogenesis is initiated and governed by cell surface integrins that bind to specific sites in the FN molecule. Recent studies identified novel integrin binding sites in FN that can also participate in FN fibril formation and in morphogenetic events during development.

Integrin-dependent and -independent functions of astrocytic fibronectin in retinal angiogenesis.

Fibronectin (FN) is a major component of the extracellular matrix and functions in cell adhesion, cell spreading and cell migration. In the retina, FN is transiently expressed and assembled on astrocytes (ACs), which guide sprouting tip cells and deposit a provisional matrix for sprouting angiogenesis. The precise function of FN in retinal angiogenesis is largely unknown. Using genetic tools, we show that astrocytes are the major source of cellular FN during angiogenesis in the mouse retina. Deletion of astrocytic FN reduces radial endothelial migration during vascular plexus formation in a gene dose-dependent manner. This effect correlates with reduced VEGF receptor 2 and PI3K/AKT signalling, and can be mimicked by selectively inhibiting VEGF-A binding to FN through intraocular injection of blocking peptides. By contrast, AC-specific replacement of the integrin-binding RGD sequence with FN-RGE or endothelial deletion of itga5 shows little effect on migration and PI3K/AKT signalling, but impairs filopodial alignment along AC processes, suggesting that FN-integrin α5ß1 interaction is involved in filopodial adhesion to the astrocytic matrix. AC FN shares its VEGF-binding function and cell-surface distribution with heparan-sulfate (HS), and genetic deletion of both FN and HS together greatly enhances the migration defect, indicating a synergistic function of FN and HS in VEGF binding. We propose that in vivo the VEGF-binding properties of FN and HS promote directional tip cell migration, whereas FN integrin-binding functions to support filopodia adhesion to the astrocytic migration template.

Shaping Oncogenic Microenvironments: Contribution of Fibronectin.

The extracellular matrix (ECM) is a complex network of proteins and glycans, dynamically remodeled and specifically tailored to the structure/function of each organ. The malignant transformation of cancer cells is determined by both cell intrinsic properties, such as mutations, and extrinsic variables, such as the mixture of surrounding cells in the tumor microenvironment and the biophysics of the ECM. During cancer progression, the ECM undergoes extensive remodeling, characterized by disruption of the basal lamina, vascular endothelial cell invasion, and development of fibrosis in and around the tumor cells resulting in increased tissue stiffness. This enhanced rigidity leads to aberrant mechanotransduction and further malignant transformation potentiating the de-differentiation, proliferation and invasion of tumor cells. Interestingly, this fibrotic microenvironment is primarily secreted and assembled by non-cancerous cells. Among them, the cancer-associated fibroblasts (CAFs) play a central role. CAFs massively produce fibronectin together with type I collagen. This review delves into the primary interactions and signaling pathways through which fibronectin can support tumorigenesis and metastasis, aiming to provide critical molecular insights for better therapy response prediction.

Profilin1 regulates sternum development and endochondral bone formation.

Bone development is a dynamic process that requires cell motility and morphological adaptation under the control of actin cytoskeleton. This actin cytoskeleton system is regulated by critical modulators including actin-binding proteins. Among them, profilin1 (Pfn1) is a key player to control actin fiber structure, and it is involved in a number of cellular activities such as migration. During the early phase of body development, skeletal stem cells and osteoblastic progenitor cells migrate to form initial rudiments for future skeletons. During this migration, these cells extend their process based on actin cytoskeletal rearrangement to locate themselves in an appropriate location within microenvironment. However, the role of Pfn1 in regulation of mesenchymal progenitor cells (MPCs) during skeletal development is incompletely understood. Here we examined the role of Pfn1 in skeletal development using a genetic ablation of Pfn1 in MPCs by using Prx1-Cre recombinase. We found that Pfn1 deficiency in MPCs caused complete cleft sternum. Notably, Pfn1-deficient mice exhibited an absence of trabecular bone in the marrow space of appendicular long bone. This phenotype is location-specific, as Pfn1 deficiency did not largely affect osteoblasts in cortical bone. Pfn1 deficiency also suppressed longitudinal growth of long bone. In vitro, Pfn1 deficiency induced retardation of osteoblastic cell migration. These observations revealed that Pfn1 is a critical molecule for the skeletal development, and this could be at least in part associated with the retardation of cell migration.

Profilin 1 is required for abscission during late cytokinesis of chondrocytes.

Profilins are key factors for dynamic rearrangements of the actin cytoskeleton. However, the functions of profilins in differentiated mammalian cells are uncertain because profilin deficiency is early embryonic lethal for higher eukaryotes. To examine profilin function in chondrocytes, we disrupted the profilin 1 gene in cartilage (Col2pfn1). Homozygous Col2pfn1 mice develop progressive chondrodysplasia caused by disorganization of the growth plate and defective chondrocyte cytokinesis, indicated by the appearance of binucleated cells. Surprisingly, Col2pfn1 chondrocytes assemble and contract actomyosin rings normally during cell division; however, they display defects during late cytokinesis as they frequently fail to complete abscission due to their inability to develop strong traction forces. This reduced force generation results from an impaired formation of lamellipodia, focal adhesions and stress fibres, which in part could be linked to an impaired mDia1-mediated actin filament elongation. Neither an actin nor a poly-proline binding-deficient profilin 1 is able to rescue the defects. Taken together, our results demonstrate that profilin 1 is not required for actomyosin ring formation in dividing chondrocytes but necessary to generate sufficient force for abscission during late cytokinesis.

Integrins, anchors and signal transducers of hematopoietic stem cells during development and in adulthood.

Hematopoietic stem cells (HSCs), the apex of the hierarchically organized blood cell production system, are generated in the yolk sac, aorta-gonad-mesonephros region and placenta of the developing embryo. To maintain life-long hematopoiesis, HSCs emigrate from their site of origin and seed in distinct microenvironments, called niches, of fetal liver and bone marrow where they receive supportive signals for self-renewal, expansion and production of hematopoietic progenitor cells (HPCs), which in turn orchestrate the production of the hematopoietic effector cells. The interactions of hematopoietic stem and progenitor cells (HSPCs) with niche components are to a large part mediated by the integrin superfamily of adhesion molecules. Here, we summarize the current knowledge regarding the functional properties of integrins and their activators, Talin-1 and Kindlin-3, for HSPC generation, function and fate decisions during development and in adulthood. In addition, we discuss integrin-mediated mechanosensing for HSC-niche interactions, ex vivo protocols aimed at expanding HSCs for therapeutic use, and recent approaches targeting the integrin-mediated adhesion in leukemia-inducing HSCs in their protecting, malignant niches.

The Role of the Fibronectin Synergy Site for Skin Wound Healing.

Skin is constantly exposed to injuries that are repaired with different outcomes, either regeneration or scarring. Scars result from fibrotic processes modulated by cellular physical forces transmitted by integrins. Fibronectin (FN) is a major component in the provisional matrix assembled to repair skin wounds. FN enables cell adhesion binding of α5ß1/αIIbß3 and αv-class integrins to an RGD-motif. An additional linkage for α5/αIIb is the synergy site located in close proximity to the RGD motif. The mutation to impair the FN synergy region (Fn1syn/syn) demonstrated that its absence permits complete development. However, only with the additional engagement to the FN synergy site do cells efficiently resist physical forces. To test how the synergy site-mediated adhesion affects the course of wound healing fibrosis, we used a mouse model of skin injury and in-vitro migration studies with keratinocytes and fibroblasts on FNsyn. The loss of FN synergy site led to normal re-epithelialization caused by two opposing migratory defects of activated keratinocytes and, in the dermis, induced reduced fibrotic responses, with lower contents of myofibroblasts and FN deposition and diminished TGF-ß1-mediated cell signalling. We demonstrate that weakened α5ß1-mediated traction forces on FNsyn cause reduced TGF-ß1 release from its latent complex.

α-parvin is required for epidermal morphogenesis, hair follicle development and basal keratinocyte polarity.

Epidermal morphogenesis and hair follicle (HF) development depend on the ability of keratinocytes to adhere to the basement membrane (BM) and migrate along the extracellular matrix. Integrins are cell-matrix receptors that control keratinocyte adhesion and migration, and are recognized as major regulators of epidermal homeostasis. How integrins regulate the behavior of keratinocytes during epidermal morphogenesis remains insufficiently understood. Here, we show that α-parvin (α-pv), a focal adhesion protein that couples integrins to actin cytoskeleton, is indispensable for epidermal morphogenesis and HF development. Inactivation of the murine α-pv gene in basal keratinocytes results in keratinocyte-BM detachment, epidermal thickening, ectopic keratinocyte proliferation and altered actin cytoskeleton polarization. In vitro, α-pv-null keratinocytes display reduced adhesion to BM matrix components, aberrant spreading and stress fibers formation, and impaired directed migration. Together, our data demonstrate that α-pv controls epidermal homeostasis by facilitating integrin-mediated adhesion and actin cytoskeleton organization in keratinocytes.

αv-Class integrin binding to fibronectin is solely mediated by RGD and unaffected by an RGE mutation.

Fibronectin (FN) is an essential glycoprotein of the extracellular matrix; binds integrins, syndecans, collagens, and growth factors; and is assembled by cells into complex fibrillar networks. The RGD motif in FN facilitates cell binding- and fibrillogenesis through binding to α5ß1 and αv-class integrins. However, whether RGD is the sole binding site for αv-class integrins is unclear. Most notably, substituting aspartate with glutamate (RGE) was shown to eliminate integrin binding in vitro, while mouse genetics revealed that FNRGE preserves αv-class integrin binding and fibrillogenesis. To address this conflict, we employed single-cell force spectroscopy, engineered cells, and RGD motif-deficient mice (Fn1ΔRGD/ΔRGD) to search for additional αv-class integrin-binding sites. Our results demonstrate that α5ß1 and αv-class integrins solely recognize the FN-RGD motif and that αv-class, but not α5ß1, integrins retain FN-RGE binding. Furthermore, Fn1ΔRGD/ΔRGD tissues and cells assemble abnormal and dysfunctional FNΔRGD fibrils in a syndecan-dependent manner. Our data highlight the central role of FN-RGD and the functionality of FN-RGE for αv-class integrins.

Material-driven fibronectin assembly rescues matrix defects due to mutations in collagen IV in fibroblasts.

Basement membranes (BMs) are specialised extracellular matrices that provide structural support to tissues as well as influence cell behaviour and signalling. Mutations in COL4A1/COL4A2, a major BM component, cause a familial form of eye, kidney and cerebrovascular disease, including stroke, while common variants in these genes are a risk factor for intracerebral haemorrhage in the general population. These phenotypes are associated with matrix defects, due to mutant protein incorporation in the BM and/or its absence by endoplasmic reticulum (ER) retention. However, the effects of these mutations on matrix stiffness, the contribution of the matrix to the disease mechanism(s) and its effects on the biology of cells harbouring a collagen IV mutation remain poorly understood. To shed light on this, we employed synthetic polymer biointerfaces, poly(ethyl acrylate) (PEA) and poly(methyl acrylate) (PMA) coated with ECM proteins laminin or fibronectin (FN), to generate controlled microenvironments and investigate their effects on the cellular phenotype of primary fibroblasts harbouring a COL4A2+/G702D mutation. FN nanonetworks assembled on PEA induced increased deposition and assembly of collagen IV in COL4A2+/G702D cells, which was associated with reduced ER size and enhanced levels of protein chaperones such as BIP, suggesting increased protein folding capacity of the cell. FN nanonetworks on PEA also partially rescued the reduced stiffness of the deposited matrix and cells, and enhanced cell adhesion through increased actin-myosin contractility, effectively rescuing some of the cellular phenotypes associated with COL4A1/4A2 mutations. The mechanism by which FN nanonetworks enhanced the cell phenotype involved integrin ß1-mediated signalling. Collectively, these results suggest that biomaterials and enhanced integrin signalling via assembled FN are able to shape the matrix and cellular phenotype of the COL4A2+/G702D mutation in patient-derived cells.

Profilin 1 delivery tunes cytoskeletal dynamics toward CNS axon regeneration.

After trauma, regeneration of adult CNS axons is abortive, causing devastating neurologic deficits. Despite progress in rehabilitative care, there is no effective treatment that stimulates axonal growth following injury. Using models with different regenerative capacities, followed by gain- and loss-of-function analysis, we identified profilin 1 (Pfn1) as a coordinator of actin and microtubules (MTs), powering axonal growth and regeneration. In growth cones, Pfn1 increased actin retrograde flow, MT growth speed, and invasion of filopodia by MTs, orchestrating cytoskeletal dynamics toward axonal growth. In vitro, active Pfn1 promoted MT growth in a formin-dependent manner, whereas localization of MTs to growth cone filopodia was facilitated by direct MT binding and interaction with formins. In vivo, Pfn1 ablation limited regeneration of growth-competent axons after sciatic nerve and spinal cord injury. Adeno-associated viral (AAV) delivery of constitutively active Pfn1 to rodents promoted axonal regeneration, neuromuscular junction maturation, and functional recovery of injured sciatic nerves, and increased the ability of regenerating axons to penetrate the inhibitory spinal cord glial scar. Thus, we identify Pfn1 as an important regulator of axonal regeneration and suggest that AAV-mediated delivery of constitutively active Pfn1, together with the identification of modulators of Pfn1 activity, should be considered to treat the injured nervous system.

Integrin activation by talin, kindlin and mechanical forces.

Integrins are the major family of adhesion molecules that mediate cell adhesion to the extracellular matrix. They are essential for embryonic development and influence numerous diseases, including inflammation, cancer cell invasion and metastasis. In this Perspective, we discuss the current understanding of how talin, kindlin and mechanical forces regulate integrin affinity and avidity, and how integrin inactivators function in this framework.

Simultaneous Boron Ion-Channel/Growth Factor Receptor Activation for Enhanced Vascularization.

Boron ion is essential in metabolism and its concentration is regulated by ion-channel NaBC1. NaBC1 mutations cause corneal dystrophies such as Harboyan syndrome. Here a 3D molecular model for NaBC1 is proposed and it is shown that simultaneous stimulation of NaBC1 and vascular endothelial growth factor receptors (VEGFR) promotes angiogenesis in vitro and in vivo with ultralow concentrations of VEGF. Human umbilical vein endothelial cells' (HUVEC) organization into tubular structures is shown to be indicative of vascularization potential. Enhanced cell sprouting is found only in the presence of VEGF and boron, the effect abrogated after blocking NaBC1. It is demonstrated that stimulated NaBC1 promotes angiogenesis via PI3k-independent pathways and that α5 ß1 /αv ß3 integrin binding is not essential to enhanced HUVEC organization. A novel vascularization mechanism that involves crosstalk and colocalization between NaBC1 and VEGFR receptors is described. This has important translational consequences; just by administering boron, taking advantage of endogenous VEGF, in vivo vascularization is shown in a chorioallantoic membrane assay.

Profilin 1 Negatively Regulates Osteoclast Migration in Postnatal Skeletal Growth, Remodeling, and Homeostasis in Mice.

Profilin 1 (Pfn1), a regulator of actin polymerization, controls cell movement in a context-dependent manner. Pfn1 supports the locomotion of most adherent cells by assisting actin-filament elongation, as has been shown in skeletal progenitor cells in our previous study. However, because Pfn1 has also been known to inhibit migration of certain cells, including T cells, by suppressing branched-end elongation of actin filaments, we hypothesized that its roles in osteoclasts may be different from that of osteoblasts. By investigating the osteoclasts in culture, we first verified that Pfn1-knockdown (KD) enhances bone resorption in preosteoclastic RAW264.7 cells, despite having a comparable number and size of osteoclasts. Pfn1-KD in bone marrow cells showed similar results. Mechanistically, Pfn1-KD osteoclasts appeared more mobile than in controls. In vivo, the osteoclast-specific conditional Pfn1-deficient mice (Pfn1-cKO) by CathepsinK-Cre driver demonstrated postnatal skeletal phenotype, including dwarfism, craniofacial deformities, and long-bone metaphyseal osteolytic expansion, by 8 weeks of age. Metaphyseal and diaphyseal femurs were drastically expanded with suppressed trabecular bone mass as indicated by µCT analysis. Histologically, TRAP-positive osteoclasts were increased at endosteal metaphysis to diaphysis of Pfn1-cKO mice. The enhanced movement of Pfn1-cKO osteoclasts in culture was associated with a slight increase in cell size and podosome belt length, as well as an increase in bone-resorbing activity. Our study, for the first time, demonstrated that Pfn1 has critical roles in inhibiting osteoclast motility and bone resorption, thereby contributing to essential roles in postnatal skeletal homeostasis. Our study also provides novel insight into understanding skeletal deformities in human disorders.