The NADPH oxidase 2 subunit p47phox binds to the WAVE regulatory complex and p22phox in a mutually exclusive manner.

The actin cytoskeleton and reactive oxygen species (ROS) both play crucial roles in various cellular processes. Previous research indicated a direct interaction between two key components of these systems: the WAVE1 subunit of the WAVE regulatory complex (WRC), which promotes actin polymerization and the p47phox subunit of the NADPH oxidase 2 complex (NOX2), which produces ROS. Here, using carefully characterized recombinant proteins, we find that activated p47phox uses its dual Src homology 3 domains to bind to multiple regions within the WAVE1 and Abi2 subunits of the WRC, without altering WRC's activity in promoting Arp2/3-mediated actin polymerization. Notably, contrary to previous findings, p47phox uses the same binding pocket to interact with both the WRC and the p22phox subunit of NOX2, albeit in a mutually exclusive manner. This observation suggests that when activated, p47phox may separately participate in two distinct processes: assembling into NOX2 to promote ROS production and engaging with WRC to regulate the actin cytoskeleton.

Identification of New Markers of Angiogenic Sprouting Using Transcriptomics: New Role for RND3.

BACKGROUND: New blood vessel formation requires endothelial cells to transition from a quiescent to an invasive phenotype. Transcriptional changes are vital for this switch, but a comprehensive genome-wide approach focused exclusively on endothelial cell sprout initiation has not been reported. METHODS: Using a model of human endothelial cell sprout initiation, we developed a protocol to physically separate cells that initiate the process of new blood vessel formation (invading cells) from noninvading cells. We used this model to perform multiple transcriptomics analyses from independent donors to monitor endothelial gene expression changes. RESULTS: Single-cell population analyses, single-cell cluster analyses, and bulk RNA sequencing revealed common transcriptomic changes associated with invading cells. We also found that collagenase digestion used to isolate single cells upregulated the Fos proto-oncogene transcription factor. Exclusion of Fos proto-oncogene expressing cells revealed a gene signature consistent with activation of signal transduction, morphogenesis, and immune responses. Many of the genes were previously shown to regulate angiogenesis and included multiple tip cell markers. Upregulation of SNAI1 (snail family transcriptional repressor 1), PTGS2 (prostaglandin synthase 2), and JUNB (JunB proto-oncogene) protein expression was confirmed in invading cells, and silencing JunB and SNAI1 significantly reduced invasion responses. Separate studies investigated rounding 3, also known as RhoE, which has not yet been implicated in angiogenesis. Silencing rounding 3 reduced endothelial invasion distance as well as filopodia length, fitting with a pathfinding role for rounding 3 via regulation of filopodial extensions. Analysis of in vivo retinal angiogenesis in Rnd3 heterozygous mice confirmed a decrease in filopodial length compared with wild-type littermates. CONCLUSIONS: Validation of multiple genes, including rounding 3, revealed a functional role for this gene signature early in the angiogenic process. This study expands the list of genes associated with the acquisition of a tip cell phenotype during endothelial cell sprout initiation.

Annexin A2 modulates phospholipid membrane composition upstream of Arp2 to control angiogenic sprout initiation.

The intersection of protein and lipid biology is of growing importance for understanding how cells address structural challenges during adhesion and migration. While protein complexes engaged with the cytoskeleton play a vital role, support from the phospholipid membrane is crucial for directing localization and assembly of key protein complexes. During angiogenesis, dramatic cellular remodeling is necessary for endothelial cells to shift from a stable monolayer to invasive structures. However, the molecular dynamics between lipids and proteins during endothelial invasion are not defined. Here, we utilized cell culture, immunofluorescence, and lipidomic analyses to identify a novel role for the membrane binding protein Annexin A2 (ANXA2) in modulating the composition of specific membrane lipids necessary for cortical F-actin organization and adherens junction stabilization. In the absence of ANXA2, there is disorganized cortical F-actin, reduced junctional Arp2, excess sprout initiation, and ultimately failed sprout maturation. Furthermore, we observed reduced filipin III labeling of membrane cholesterol in cells with reduced ANXA2, suggesting there is an alteration in phospholipid membrane dynamics. Lipidomic analyses revealed that 42 lipid species were altered with loss of ANXA2, including an accumulation of phosphatidylcholine (16:0_16:0). We found that supplementation of phosphatidylcholine (16:0_16:0) in wild-type endothelial cells mimicked the ANXA2 knock-down phenotype, indicating that ANXA2 regulated the phospholipid membrane upstream of Arp2 recruitment and organization of cortical F-actin. Altogether, these data indicate a novel role for ANXA2 in coordinating events at endothelial junctions needed to initiate sprouting and show that proper lipid modulation is a critical component of these events.

MicroRNA-150 (miR-150) and Diabetic Retinopathy: Is miR-150 Only a Biomarker or Does It Contribute to Disease Progression?

Diabetic retinopathy (DR) is a chronic disease associated with diabetes mellitus and is a leading cause of visual impairment among the working population in the US. Clinically, DR has been diagnosed and treated as a vascular complication, but it adversely impacts both neural retina and retinal vasculature. Degeneration of retinal neurons and microvasculature manifests in the diabetic retina and early stages of DR. Retinal photoreceptors undergo apoptosis shortly after the onset of diabetes, which contributes to the retinal dysfunction and microvascular complications leading to vision impairment. Chronic inflammation is a hallmark of diabetes and a contributor to cell apoptosis, and retinal photoreceptors are a major source of intraocular inflammation that contributes to vascular abnormalities in diabetes. As the levels of microRNAs (miRs) are changed in the plasma and vitreous of diabetic patients, miRs have been suggested as biomarkers to determine the progression of diabetic ocular diseases, including DR. However, few miRs have been thoroughly investigated as contributors to the pathogenesis of DR. Among these miRs, miR-150 is downregulated in diabetic patients and is an endogenous suppressor of inflammation, apoptosis, and pathological angiogenesis. In this review, how miR-150 and its downstream targets contribute to diabetes-associated retinal degeneration and pathological angiogenesis in DR are discussed. Currently, there is no effective treatment to stop or reverse diabetes-caused neural and vascular degeneration in the retina. Understanding the molecular mechanism of the pathogenesis of DR may shed light for the future development of more effective treatments for DR and other diabetes-associated ocular diseases.

The ß1-integrin plays a key role in LEC invasion in an optimized 3-D collagen matrix model.

Lymphangiogenesis, or formation of new lymphatic vessels, is a tightly regulated process that is controlled by growth factor signaling and biomechanical cues. Lymphatic endothelial cells (LECs) undergo remodeling, migration, and proliferation to invade the surrounding extracellular matrix (ECM) during both physiological and pathological lymphangiogenesis. This study optimized conditions for an in vitro three-dimensional (3-D) collagen-based model that induced LEC invasion and recapitulated physiological formation of lymphatic capillaries with lumens. Invasion of LECs was enhanced in the presence of sphingosine 1-phosphate (S1P). Effects of various known lymphangiogenic factors, vascular endothelial growth factor (VEGF)-A, basic fibroblast growth factor (bFGF), interleukin (IL)-8, and hepatocyte growth factor (HGF), were tested on LEC sprout formation synergistically with VEGF-C. Several of these growth factors significantly enhanced LEC invasion, and synergistic effects of some of these further enhanced the sprouting density and lumen volume. To determine the contribution of specific ECM components, we analyzed the expression of different integrin subunits. Basal expressions of the integrin α5- and integrin ß1-subunits were high in LECs. The addition of fibronectin, which mediates cellular responses through these integrins, enhanced LEC sprouting density and sprout length dose-dependently. siRNA-mediated knockdown of the integrin ß1-subunit suppressed LEC invasion and also inhibited VEGF receptor (VEGFR)3 and ERK activation. Furthermore, exposing LECs to the inflammatory mediator lipopolysaccharide (LPS) inhibited sprouting. This optimized model for LEC invasion includes S1P, VEGF-C, and fibronectin within a 3-D collagen matrix, along with VEGF-C, VEGF-A, bFGF, and HGF in the culture medium, and provides a useful tool to investigate the functional effect of various lymphangiogenic factors and inhibitors.

Transforming growth factor beta signaling and decidual integrity in mice†.

Transforming growth factor beta (TGFß) signaling regulates multifaceted reproductive processes. It has been shown that the type 1 receptor of TGFß (TGFBR1) is indispensable for female reproductive tract development, implantation, placental development, and fertility. However, the role of TGFß signaling in decidual development and function remains poorly defined. Our objective is to determine the impact of uterine-specific deletion of Tgfbr1 on decidual integrity, with a focus on the cellular and molecular properties of the decidua during development. Our results show that the developmental dynamics of the decidua is altered in TGFBR1 conditionally depleted uteri from embryonic day (E) 5.5 to E8.5, substantiated by downregulation of genes associated with inflammatory responses and uterine natural killer cell abundance, reduced presence of nondecidualized fibroblasts in the antimesometrial region, and altered decidual cell development. Notably, conditional ablation of TGFBR1 results in the formation of decidua containing more abundant alpha smooth muscle actin (ACTA2)-positive cells at the peripheral region of the antimesometrial side versus controls at E6.5-E8.5. This finding is corroborated by upregulation of a subset of smooth muscle marker genes in Tgfbr1 conditionally deleted decidua at E6.5 and E8.5. Moreover, increased cell proliferation and enhanced decidual ERK1/2 signaling were found in Tgfbr1 conditional knockout mice upon decidual regression. In summary, conditional ablation of TGFBR1 in the uterus profoundly impacts the cellular and molecular properties of the decidua. Our results suggest that TGFBR1 in uterine epithelial and stromal compartments is important for the integrity of the decidua, a transient but crucial structure that supports embryo development.

Enhancer of Zeste 2 Polycomb Repressive Complex 2 Subunit Is Required for Uterine Epithelial Integrity.

Normal proliferation and differentiation of uterine epithelial cells are critical for uterine development and function. Enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2), a core component of polycomb repressive complexes 2, possesses histone methyltransferase activity that catalyzes the trimethylation of lysine 27 of histone H3. EZH2 has been involved in epithelial-mesenchymal transition, a key event in development and carcinogenesis. However, its role in uterine epithelial cell function remains unknown. To determine the role of uterine EZH2, Ezh2 was conditionally deleted using progesterone receptor Cre recombinase, which is expressed in both epithelial and mesenchymal compartments of the uterus. Loss of EZH2 promoted stratification of uterine epithelium, an uncommon and detrimental event in the uterus. The abnormal epithelium expressed basal cell markers, including tumor protein 63, cytokeratin 5 (KRT5), KRT6A, and KRT14. These results suggest that EZH2 serves as a guardian of uterine epithelial integrity, partially via inhibiting the differentiation of basal-like cells and preventing epithelial stratification. The observed epithelial abnormality was accompanied by fertility defects, altered uterine growth and function, and the development of endometrial hyperplasia. Thus, the Ezh2 conditional knockout mouse model may be useful to explore mechanisms that regulate endometrial homeostasis and uterine function.

OPN binds alpha V integrin to promote endothelial progenitor cell incorporation into vasculature.

Angiogenesis is fundamental to the expansion of the placental vasculature during pregnancy. Integrins are associated with vascular formation; and osteopontin is a candidate ligand for integrins to promote angiogenesis. Endothelial progenitor cells (EPCs) are released from bone marrow into the blood and incorporate into newly vascularized tissue where they differentiate into mature endothelium. Results of studies in women suggest that EPCs may play an important role in maintaining placental vascular integrity during pregnancy, although little is known about how EPCs are recruited to these tissues. Our goal was to determine the αv integrin mediated effects of osteopontin on EPC adhesion and incorporation into angiogenic vascular networks. EPCs were isolated from 6 h old piglets. RT-PCR revealed that EPCs initially had a monocyte-like phenotype in culture that became more endothelial-like with cell passage. Immunofluorescence microscopy confirmed that the EPCs express platelet endothelial cell adhesion molecule, vascular endothelial cadherin, and von Willebrand factor. When EPCs were cultured on OPN-coated slides, the αv integrin subunit was observed in focal adhesions at the basal surface of EPCs. Silencing of αv integrin reduced EPC binding to OPN and focal adhesion assembly. In vitro siRNA knockdown in EPCs,demonstrated that OPN stimulates EPC incorporation into human umbilical vein endothelial cell (HUVEC) networks via αv-containing integrins. Finally, in situ hybridization and immunohistochemistry localized osteopontin near placental blood vessels. In summary, OPN binds the αv integrin subunit on EPCs to support EPC adhesion and increase EPC incorporation into angiogenic vascular networks.

Selective regulation of Notch ligands during angiogenesis is mediated by vimentin.

Notch signaling is a key regulator of angiogenesis, in which sprouting is regulated by an equilibrium between inhibitory Dll4-Notch signaling and promoting Jagged-Notch signaling. Whereas Fringe proteins modify Notch receptors and strengthen their activation by Dll4 ligands, other mechanisms balancing Jagged and Dll4 signaling are yet to be described. The intermediate filament protein vimentin, which has been previously shown to affect vascular integrity and regenerative signaling, is here shown to regulate ligand-specific Notch signaling. Vimentin interacts with Jagged, impedes basal recycling endocytosis of ligands, but is required for efficient receptor ligand transendocytosis and Notch activation upon receptor binding. Analyses of Notch signal activation by using chimeric ligands with swapped intracellular domains (ICDs), demonstrated that the Jagged ICD binds to vimentin and contributes to signaling strength. Vimentin also suppresses expression of Fringe proteins, whereas depletion of vimentin enhances Fringe levels to promote Dll4 signaling. In line with these data, the vasculature in vimentin knockout (VimKO) embryos and placental tissue is underdeveloped with reduced branching. Disrupted angiogenesis in aortic rings from VimKO mice and in endothelial 3D sprouting assays can be rescued by reactivating Notch signaling by recombinant Jagged ligands. Taken together, we reveal a function of vimentin and demonstrate that vimentin regulates Notch ligand signaling activities during angiogenesis.

Piezo1 mediates angiogenesis through activation of MT1-MMP signaling.

Angiogenesis is initiated in response to a variety of external cues, including mechanical and biochemical stimuli; however, the underlying signaling mechanisms remain unclear. Here, we investigated the proangiogenic role of the endothelial mechanosensor Piezo1. Genetic deletion and pharmacological inhibition of Piezo1 reduced endothelial sprouting and lumen formation induced by wall shear stress and proangiogenic mediator sphingosine 1-phosphate, whereas Piezo1 activation by selective Piezo1 activator Yoda1 enhanced sprouting angiogenesis. Similarly to wall shear stress, sphingosine 1-phosphate functioned by activating the Ca2+ gating function of Piezo1, which in turn signaled the activation of the matrix metalloproteinase-2 and membrane type 1 matrix metalloproteinase during sprouting angiogenesis. Studies in mice in which Piezo1 was conditionally deleted in endothelial cells demonstrated the requisite role of sphingosine 1-phosphate-dependent activation of Piezo1 in mediating angiogenesis in vivo. These results taken together suggest that both mechanical and biochemical stimuli trigger Piezo1-mediated Ca2+ influx and thereby activate matrix metalloproteinase-2 and membrane type 1 matrix metalloproteinase and synergistically facilitate sprouting angiogenesis.

Hic-5 mediates the initiation of endothelial sprouting by regulating a key surface metalloproteinase.

During angiogenesis, endothelial cells must coordinate matrix proteolysis with migration. Here, we tested whether the focal adhesion scaffold protein Hic-5 (also known as TGFB1I1) regulated endothelial sprouting in three dimensions. Hic-5 silencing reduced endothelial sprouting and lumen formation, and sprouting defects were rescued by the return of Hic-5 expression. Pro-angiogenic factors enhanced colocalization and complex formation between membrane type-1 matrix metalloproteinase (MT1-MMP, also known as MMP14) and Hic-5, but not between paxillin and MT1-MMP. The LIM2 and LIM3 domains of Hic-5 were necessary and sufficient for Hic-5 to form a complex with MT1-MMP. The degree of interaction between MT1-MMP and Hic-5 and the localization of the complex within detergent-resistant membrane fractions were enhanced during endothelial sprouting, and Hic-5 depletion lowered the surface levels of MT1-MMP. In addition, we observed that loss of Hic-5 partially reduced complex formation between MT1-MMP and focal adhesion kinase (FAK, also known as PTK2), suggesting that Hic-5 bridges MT1-MMP and FAK. Finally, Hic-5 LIM2-LIM3 deletion mutants reduced sprout initiation. Hic-5, MT1-MMP and FAK colocalized in angiogenic vessels during porcine pregnancy, supporting that this complex assembles during angiogenesis in vivo. Collectively, Hic-5 appears to enhance complex formation between MT1-MMP and FAK in activated endothelial cells, which likely coordinates matrix proteolysis and cell motility.

Establishment of a three-dimensional model to study human uterine angiogenesis.

STUDY QUESTION: Can primary human uterine microvascular endothelial cells (UtMVECs) be used as a model to study uterine angiogenic responses in vitro that are relevant in pregnancy? SUMMARY ANSWER: UtMVECs demonstrated angiogenic responses when stimulated with proangiogenic factors, including sphingosine 1-phosphate (S1P), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), physiological levels of wall shear stress (WSS), human chorionic gonadotropin (hCG) and various combinations of estrogen and progesterone. WHAT IS KNOWN ALREADY: During sprouting angiogenesis, signaling from growth factors and cytokines induces a monolayer of quiescent endothelial cells (ECs) lining the vasculature to degrade the extracellular matrix and invade the surrounding tissue to form new capillaries. During pregnancy and the female reproductive cycle, the uterine endothelium becomes activated and undergoes sprouting angiogenesis to increase the size and number of blood vessels in the endometrium. STUDY DESIGN, SIZE, DURATION: The study was designed to examine the angiogenic potential of primary human UtMVECs using the well-characterized human umbilical vein EC (HUVEC) line as a control to compare angiogenic potential. ECs were seeded onto three-dimensional (3D) collagen matrices, supplemented with known proangiogenic stimuli relevant to pregnancy and allowed to invade for 24 h. Sprouting responses were analyzed using manual and automated methods for quantification. PARTICIPANTS/MATERIALS, SETTING, METHODS: RT-PCR, Western blot analysis and immunostaining were used to characterize UtMVECs. Angiogenic responses were examined using 3D invasion assays. Western blotting was used to confirm signaling responses after proangiogenic lipid, pharmacological inhibitor, and recombinant lentiviral treatments. All experiments were repeated at least three times. MAIN RESULTS AND THE ROLE OF CHANCE: After ensuring that UtMVECs expressed the proper endothelial markers, we found that UtMVECs invade 3D collagen matrices dose-dependently in response to known proangiogenic stimuli (e.g. S1P, VEGF, bFGF, hCG, estrogen, progesterone and WSS) present during early pregnancy. Invasion responses were positively correlated with phosphorylation of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) and p42/p44 mitogen-activated protein kinase (ERK). Inhibition of these second messengers significantly impaired sprouting (P < 0.01). Gene silencing of membrane type 1-matrix metalloproteinase using multiple approaches completely abrogated sprouting (P < 0.001). Finally, UtMVECs displayed a unique ability to undergo sprouting in response to hCG, and combined estrogen and progesterone treatment. LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: The study of uterine angiogenesis in vitro has limitations and any findings many not fully represent the in vivo state. However, these experiments do provide evidence for the ability of UtMVECs to be used in functional sprouting assays in a 3D environment, stimulated by physiological factors that are produced locally within the uterus during early pregnancy. WIDER IMPLICATIONS OF THE FINDINGS: We show that UtMVECs can be used reliably to investigate how growth factors, hormones, lipids and other factors, such as flow, affect angiogenesis in the uterus. STUDY FUNDING/COMPETING INTERESTS: This work was supported by NIH award HL095786 to K.J.B. The authors have no conflicts of interest.

ITGAV (alpha v integrins) bind SPP1 (osteopontin) to support trophoblast cell adhesion.

Attachment of the conceptus trophoblast (Tr) to the uterine luminal epithelium (LE) is critical for successful implantation. This study determined whether alpha v (av) integrins (ITGAV) directly mediate porcine trophoblast cell adhesion to secreted phosphoprotein 1 (SPP1, also known as osteopontin (OPN)) and examined the temporal/spatial expression of ITGAV, beta 3 (b3, ITGB3) and beta 6 (b6, ITGB6) integrin subunits, and SPP1, at the uterine-placental interface of pigs. Knockdown of ITGAV in porcine Tr (pTr2) cells by siRNA reduced pTr2 attachment to SPP1. In situ hybridization confirmed the presence of ITGAV, ITGB3 and ITGB6 mRNAs in uterine LE and conceptus Tr between Days 9 and 60 of gestation, with no change in the magnitude of expression over the course of pregnancy. Exogenous E2 or P4 did not affect ITGAV, ITGB3 and ITGB6 mRNA expression in the uteri of ovariectomized gilts. Immunofluorescence identified ITGAV, ITGB3 and SPP1 proteins in large aggregates at the uterine LE-placental Tr/chorion interface on Day 25, but aggregates were no longer observed by Day 50 of gestation. These results are the first to directly demonstrate that pTr2 cells engage ITGAV-containing integrin receptors to adhere to SPP1 and suggest that mechanical forces generated by tethering elongating conceptuses to uterine LE leads to assembly of focal adhesions containing ITGAV and SPP1; however, as placentation progresses, subsequent folding/interdigitation at the uterine-placental interface disperses mechanical forces resulting in the loss of focal adhesions.

Separating full-length protein from aggregating proteolytic products using filter flow-through purification.

Separation of full-length protein from proteolytic products is challenging, since the properties used to isolate the protein can also be present in proteolytic products. Many separation techniques risk non-specific protein adhesion and/or require a lot of time, enabling continued proteolysis and aggregation after lysis. We demonstrate that proteolytic products aggregate for two different proteins. As a result, full-length protein can be rapidly separated from these fragments by filter flow-through purification, resulting in a substantial protein purity enhancement. This rapid approach is likely to be useful for intrinsically disordered proteins, whose repetitive sequence composition and flexible nature can facilitate aggregation.

Functionalization of Ultrabithorax Materials with Vascular Endothelial Growth Factor Enhances Angiogenic Activity.

Successful design of tissue engineering scaffolds must include the ability to stimulate vascular development by incorporating angiogenic growth factors. Current approaches can allow diffusion of growth factors, incorporate active factors randomly, or can leave residual toxins. We addressed these problems by genetically fusing the gene encoding Vascular Endothelial Growth Factor (VEGF) with the Ultrabithorax (Ubx) gene to produce fusion proteins capable of self-assembly into materials. We demonstrate that VEGF-Ubx materials enhance human endothelial cell migration, prolong cell survival, and dose-dependently activate the VEGF signaling pathway. VEGF-Ubx fibers attract outgrowing sprouts in an aortic ring assay and induce vessel formation in a chicken embryo chorioallantoic membrane (CAM) assay. Collectively, these results demonstrate that the activity of VEGF remains intact in Ubx materials. This approach could provide an inexpensive and facile mechanism to stimulate and pattern angiogenesis.

Fluid shear stress promotes proprotein convertase-dependent activation of MT1-MMP.

During angiogenesis, endothelial cells (ECs(1)) initiate new blood vessel growth and invade into the extracellular matrix (ECM). Membrane type-1 matrix metalloproteinase (MT1-MMP) facilitates this process and translocates to the plasma membrane following activation to promote ECM cleavage. The N-terminal pro-domain within MT1-MMP must be processed for complete activity of the proteinase. This study investigated whether MT1-MMP activation was altered by sphingosine 1-phosphate (S1P) and wall shear stress (WSS), which combine to stimulate EC invasion in three dimensional (3D) collagen matrices. MT1-MMP was activated rapidly and completely by WSS but not S1P. Proprotein convertases (PCs) promoted MT1-MMP processing, prompting us to test whether WSS or S1P treatments increased PC activity. Like MT1-MMP, PC activity increased with WSS, while S1P had no effect. A pharmacological PC inhibitor completely blocked S1P- and WSS-induced EC invasion and MT1-MMP translocation to the plasma membrane. Further, a recombinant PC inhibitor reduced MT1-MMP activation and decreased lumen formation in invading ECs, a process known to be controlled by MT1-MMP. Thus, we conclude that PC and MT1-MMP activation are mechanosensitive events that are required for EC invasion into 3D collagen matrices.

Identification of multiple dityrosine bonds in materials composed of the Drosophila protein Ultrabithorax.

The recombinant protein Ultrabithorax (Ubx), a Drosophila melanogaster Hox transcription factor, self-assembles into biocompatible materials in vitro that are remarkably extensible and strong. Here, we demonstrate that the strength of Ubx materials is due to intermolecular dityrosine bonds. Ubx materials auto-fluoresce blue, a characteristic of dityrosine, and bind dityrosine-specific antibodies. Monitoring the fluorescence of reduced Ubx fibers upon oxygen exposure reveals biphasic bond formation kinetics. Two dityrosine bonds in Ubx were identified by site-directed mutagenesis followed by measurements of fiber fluorescent intensity. One bond is located between the N-terminus and the homeodomain (Y4/Y296 or Y12/Y293), and another bond is formed by Y167 and Y240. Fiber fluorescence closely correlates with fiber strength, demonstrating that these bonds are intermolecular. To our knowledge, this is the first identification of specific residues that participate in dityrosine bonds in protein-based materials. The percentage of Ubx molecules harboring both bonds can be decreased or increased by mutagenesis, providing an additional mechanism to control the mechanical properties of Ubx materials. Duplication of tyrosine-containing motifs in Ubx increases dityrosine content in Ubx fibers, suggesting these motifs could be inserted in other self-assembling proteins to strengthen the corresponding materials.

Constitutive activation of transforming growth factor Beta receptor 1 in the mouse uterus impairs uterine morphology and function.

Despite increasing evidence pointing to the essential involvement of the transforming growth factor beta (TGFB) superfamily in reproduction, a definitive role of TGFB signaling in the uterus remains to be unveiled. In this study, we generated a gain-of-function mouse model harboring a constitutively active (CA) TGFB receptor 1 (TGFBR1), the expression of which was conditionally induced by the progesterone receptor (Pgr)-Cre recombinase. Overactivation of TGFB signaling was verified by enhanced phosphorylation of SMAD2 and increased expression of TGFB target genes in the uterus. TGFBR1 Pgr-Cre CA mice were sterile. Histological, cellular, and molecular analyses demonstrated that constitutive activation of TGFBR1 in the mouse uterus promoted formation of hypermuscled uteri. Accompanying this phenotype was the upregulation of a battery of smooth muscle genes in the uterus. Furthermore, TGFB ligands activated SMAD2/3 and stimulated the expression of a smooth muscle maker gene, alpha smooth muscle actin (ACTA2), in human uterine smooth muscle cells. Immunofluorescence microscopy identified a marked reduction of uterine glands in TGFBR1 Pgr-Cre CA mice within the endometrial compartment that contained myofibroblast-like cells. Thus, constitutive activation of TGFBR1 in the mouse uterus caused defects in uterine morphology and function, as evidenced by abnormal myometrial structure, dramatically reduced uterine glands, and impaired uterine decidualization. These results underscore the importance of a precisely controlled TGFB signaling system in establishing a uterine microenvironment conducive to normal development and function.

Proteomic profiling of endothelial invasion revealed receptor for activated C kinase 1 (RACK1) complexed with vimentin to regulate focal adhesion kinase (FAK).

Angiogenesis is critical for many physiological and pathological processes. To identify molecules relevant to angiogenesis, we performed a proteomic screen comparing invading versus non-invading endothelial cells in three-dimensional collagen matrices. We found up-regulated levels of receptor for activated C kinase 1 (RACK1) and the intermediate filament protein vimentin that correlated with increased endothelial cell invasion. Because both RACK1 and vimentin have been linked to focal adhesion kinase (FAK), we investigated whether this pathway regulated invasion. RACK1 depletion reduced invasion responses, and this was associated with attenuated activation of FAK. Knockdown of vimentin significantly decreased levels of phosphorylated and total FAK. Treatment with a pharmacological inhibitor of FAK dose-dependently reduced invasion, indicating a crucial role for FAK activity during invasion. Because RACK1 and vimentin were both up-regulated with sphingosine 1-phosphate treatment, required for invasion, and regulated FAK, we tested whether they complexed together. RACK1 complexed with vimentin, and growth factors enhanced this interaction. In addition, RACK1, vimentin, and FAK formed an intermolecular complex in invading endothelial cultures in three dimensions in response to stimulation by sphingosine 1-phosphate and growth factors. Moreover, depletion of RACK1 decreased the association of vimentin and FAK, suggesting that RACK1 was required for stabilizing vimentin-FAK interactions during sprouting. Silencing of vimentin and RACK1 decreased cell adhesion and focal contact formation. Taken together, these results demonstrate that proangiogenic signals converge to enhance expression and association of RACK1 and vimentin, which regulated FAK, resulting in successful endothelial sprout formation in three-dimensional collagen matrices.