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Glucokinase-mediated glucose signaling induces insulin secretion, proliferation, and apoptosis in pancreatic β-cells. However, the precise molecular mechanisms underlying these processes are not clearly understood. Here, we demonstrated that glucokinase activation using a glucokinase activator (GKA) significantly upregulated the expression of Fibulin-5 (Fbln5), a matricellular protein involved in matrix-cell signaling, in isolated mouse islets. The islet Fbln5 expression was induced by ambient glucose in a time- and dose-dependent manner and further enhanced by high-fat diet or the deletion of insulin receptor substrate 2 (IRS-2), whereas the GKA-induced increase in Fbln5 expression was diminished in Irs-2-deficient islets. GKA-induced Fbln5 upregulation in the islets was blunted by a glucokinase inhibitor, K ATP channel opener, Ca 2+ channel blocker and calcineurin inhibitor, while it was augmented by harmine, a dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) 1 A inhibitor. Although deletion of Fbln5 in mice had no significant effects on the glucose tolerance or β-cell functions, adenovirus-mediated Fbln5 overexpression increased glucose-stimulated insulin secretion in INS-1 rat insulinoma cells. Since the islet Fbln5 expression is regulated through a glucokinase/K ATP channel/calcineurin/nuclear factor of activated T cells (NFAT) pathway crucial for the maintenance of β-cell functions, further investigation of Fbln5 functions in the islets is warranted.

Compromise of elastic fiber integrity in connective tissues of the pelvic floor is most likely acquired through aging, childbirth-associated injury, and genetic susceptibility. Mouse models of pelvic organ prolapse demonstrate systemic deficiencies in proteins that affect elastogenesis. Prolapse, however, does not occur until several months after birth and is thereby acquired with age or after parturition. To determine the impact of compromised levels of fibulin-5 (Fbln5) during adulthood on pelvic organ support after parturition and elastase-induced injury, tissue-specific conditional knockout (cKO) mice were generated in which doxycycline (dox) treatment results in deletion of Fbln5 in cells that utilize the smooth muscle α actin promoter-driven reverse tetracycline transactivator and tetracycline responsive element-Cre recombinase (i.e., Fbln5f/f/SMA++-rtTA/Cre+, cKO). Fbln5 was decreased significantly in the vagina of cKO mice compared with dox-treated wild type or controls (Fbln5f/f/SMA++-rtTA/Cre-/-). In controls, perineal body length (PBL) and bulge increased significantly after delivery but declined to baseline values within 6-8 weeks. Although overt prolapse did not occur in cKO animals, these transient increases in PBL postpartum were amplified and, unlike controls, parturition-induced increases in PBL (and bulge) did not recover to baseline but remained significantly increased for 12 wks. This lack of recovery from parturition was associated with increased MMP-9 and nondetectable levels of Fbln5 in the postpartum vagina. This predisposition to prolapse was accentuated by injection of elastase into the vaginal wall in which overt prolapse occurred in cKO animals, but rarely in controls. Taken together, our model system in which Fbln5 is conditionally knock-downed in stromal cells of the pelvic floor results in animals that undergo normal elastogenesis during development but lose Fbln5 as adults. The results indicate that vaginal fibulin-5 during development is crucial for baseline pelvic organ support and is also important for protection and recovery from parturition- and elastase-induced prolapse.

Elevated oxidative stress is an aberration seen in many solid tumors, and exploiting this biochemical difference has the potential to enhance the efficacy of anticancer agents. Homeostasis of reactive oxygen species (ROS) is important for normal cell function, but excessive production of ROS can result in cellular toxicity, and therefore ROS levels must be balanced finely. Here, we highlight the relationship between the extracellular matrix and ROS production by reporting a novel function of the matricellular protein Fibulin-5 (Fbln5). We used genetically engineered mouse models of pancreatic ductal adenocarcinoma (PDAC) and found that mutation of the integrin-binding domain of Fbln5 led to decreased tumor growth, increased survival, and enhanced chemoresponse to standard PDAC therapies. Through mechanistic investigations, we found that improved survival was due to increased levels of oxidative stress in Fbln5-mutant tumors. Furthermore, loss of the Fbln5-integrin interaction augmented fibronectin signaling, driving integrin-induced ROS production in a 5-lipooxygenase-dependent manner. These data indicate that Fbln5 promotes PDAC progression by functioning as a molecular rheostat that modulates cell-ECM interactions to reduce ROS production, and thus tip the balance in favor of tumor cell survival and treatment-refractory disease., (©2015 American Association for Cancer Research.)

Numerous diseases have been linked to genetic mutations that lead to reduced amounts or disorganization of arterial elastic fibres. Previous work has shown that mice with reduced amounts of elastin (Eln+/-) are able to live a normal lifespan through cardiovascular adaptations, including changes in haemodynamic stresses, arterial geometry and arterial wall mechanics. It is not known if the timeline and presence of these adaptations are consistent in other mouse models of elastic fibre disease, such as those caused by the absence of fibulin-5 expression (Fbln5-/-). Adult Fbln5-/- mice have disorganized elastic fibres, decreased arterial compliance and high blood pressure. We examined mechanical behaviour of the aorta in Fbln5-/- mice through early maturation when the elastic fibres are being assembled. We found that the physiologic circumferential stretch, stress and modulus of Fbln5-/- aorta are maintained near wild-type levels. Constitutive modelling suggests that elastin contributions to the total stress are decreased, whereas collagen contributions are increased. Understanding how collagen fibre structure and mechanics compensate for defective elastic fibres to meet the mechanical requirements of the maturing aorta may help to better understand arterial remodelling in human elastinopathies., (© 2015 The Author(s) Published by the Royal Society. All rights reserved.)

uPA (urokinase-type plasminogen activator) stimulates cell migration through multiple pathways, including formation of plasmin and extracellular metalloproteinases, and binding to the uPAR (uPA receptor; also known as CD87), integrins and LRP1 (low-density lipoprotein receptor-related protein 1) which activate intracellular signalling pathways. In the present paper we report that uPA-mediated cell migration requires an interaction with fibulin-5. uPA stimulates migration of wild-type MEFs (mouse embryonic fibroblasts) (Fbln5+/+ MEFs), but has no effect on fibulin-5-deficient (Fbln5-/-) MEFs. Migration of MEFs in response to uPA requires an interaction of fibulin-5 with integrins, as MEFs expressing a mutant fibulin-5 incapable of binding integrins (Fbln(RGE/RGE) MEFs) do not migrate in response to uPA. Moreover, a blocking anti-(human β1-integrin) antibody inhibited the migration of PASMCs (pulmonary arterial smooth muscle cells) in response to uPA. Binding of uPA to fibulin-5 generates plasmin, which excises the integrin-binding N-terminal cbEGF (Ca2+-binding epidermal growth factor)-like domain, leading to loss of β1-integrin binding. We suggest that uPA promotes cell migration by binding to fibulin-5, initiating its cleavage by plasmin, which leads to its dissociation from β1-integrin and thereby unblocks the capacity of integrin to facilitate cell motility.

Tumor survival depends in part on the ability of tumor cells to transform the surrounding extracellular matrix (ECM) into an environment conducive to tumor progression. Matricellular proteins are secreted into the ECM and impact signaling pathways that are required for pro-tumorigenic activities such as angiogenesis. Fibulin-5 (Fbln5) is a matricellular protein that was recently shown to regulate angiogenesis; however, its effect on tumor angiogenesis and thus tumor growth is currently unknown. We report that the growth of pancreatic tumors and tumor angiogenesis is suppressed in Fbln5-null (Fbln5(-/-)) mice compared with wild-type (WT) littermates. Furthermore, we observed an increase in the level of reactive oxygen species (ROS) in tumors grown in Fbln5(-/-) animals. Increased ROS resulted in elevated DNA damage, increased apoptosis of endothelial cells within the tumor, and represented the underlying cause for the reduction in angiogenesis and tumor growth. In vitro, we identified a novel pathway by which Fbln5 controls ROS production through a mechanism that is dependent on beta1 integrins. These results were validated in Fbln5(RGE/RGE) mice, which harbor a point mutation in the integrin-binding RGD motif of Fbln5, preventing its interaction with integrins. Tumor growth and angiogenesis was reduced in Fbln5(RGE/RGE) mice, however treatment with an antioxidant rescued angiogenesis and elevated tumor growth to WT levels. These findings introduce a novel function for Fbln5 in the regulation of integrin-induced ROS production and establish a rationale for future studies to examine whether blocking Fbln5 function could be an effective anti-tumor strategy, alone or in combination with other therapies.

Objective: Recent findings on the role of fibulin-5 (Fbln5) have provided substantial progress in understanding the molecular mechanism of elastic fiber assembly in vitro. However, little is known about differential roles of fibulins in the elastogenesis of blood vessels. Here, we generated double knockout mice for Fbln5 and Fbln2 (termed DKO) and examined the role of fibulins-2 and -5 in development and injury response of the blood vessel wall., Methods and Results: Fibulin-2 is distinctly located in the subendothelial matrix, whereas fibulin-5 is observed throughout the vessel wall. All of the elastic laminae, including the internal elastic lamina (IEL), were severely disorganized in DKO mice, which was not observed in single knockout mice for Fbln2 or Fbln5. Furthermore, DKO vessels displayed upregulation of vascular adhesion molecules, tissue factor expression, and thrombus formation with marked dilation and thinning of the vessel wall after carotid artery ligation-injury., Conclusions: Fibulin-2 and fibulin-5 cooperatively function to form the IEL during postnatal development by directing the assembly of elastic fibers, and are responsible for maintenance of the adult vessel wall after injury. The DKO mouse will serve as a unique animal model to test the effect of vessel integrity during various pathological insults.

Two mouse models of pelvic organ prolapse have been generated recently, both of which have null mutations in genes involved in elastic fiber synthesis and assembly (fibulin 5 and lysyl oxidase-like 1). Interestingly, although these mice exhibit elastinopathies early in life, pelvic organ prolapse does not develop until later in life. In this investigation we developed and validated a tool to quantify the severity of pelvic organ prolapse in mice, and we used this tool prospectively to study the role of fibulin 5, aging, and vaginal proteases in the development of pelvic organ prolapse. The results indicate that >90% of Fbln5(-/-) mice develop prolapse by 6 mo of age, even in the absence of vaginal delivery, and that increased vaginal protease activity precedes the development of prolapse.

Elastic fibers contribute to the structural support of tissues and to the regulation of cellular behavior. Mice deficient for the fibulin-5 gene (fbln5(-/-)) were used to further elucidate the molecular mechanism of elastic fiber assembly. Major elastic fiber components were present in the skin of fbln5(-/-) mice despite a dramatic reduction of mature elastic fibers. We found that fibulin-5 preferentially bound the monomeric form of elastin through N-terminal and C-terminal elastin-binding regions and to a preexisting matrix scaffold through calcium-binding epidermal growth factor (EGF)-like (CB-EGF) domains. We further showed that adenovirus-mediated gene transfer of fbln5 was sufficient to regenerate elastic fibers and increase elastic fiber-cell connections in vivo. A mutant fibulin-5 lacking the first 28 amino acids of the first CB-EGF domain, however, was unable to rescue elastic fiber defects. Fibulin-5 thus serves as an adaptor molecule between monomeric elastin and the matrix scaffold to aid in elastic fiber assembly. These results also support the potential use of fibulin-5 as a therapeutic agent for the treatment of elastinopathies.

Transgenic mice overexpressing the calcium binding protein, S100A4/Mts1, occasionally develop severe pulmonary vascular obstructive disease. To understand what underlies this propensity, we compared the pulmonary vascular hemodynamic and structural features of S100A4/Mts1 with control C57Bl/6 mice at baseline, following a 2-week exposure to chronic hypoxia, and after 1 and 3 months "recovery" in room air. S100A4/Mts1 mice had greater right ventricular systolic pressure and right ventricular hypertrophy at baseline, which increased further with chronic hypoxia and was sustained after 3 months "recovery" in room air. These findings correlated with a heightened response to acute hypoxia and failure to vasodilate with nitric oxide or oxygen. S100A4/Mts1 mice, when compared with C57Bl/6 mice, also had impaired cardiac function judged by reduced ventricular elastance and decreased cardiac output. Despite higher right ventricular systolic pressures with chronic hypoxia, S100A4/Mts1 mice did not develop more severe PVD, but in contrast to C57Bl/6 mice, these features did not regress on return to room air. Microarray analysis of lung tissue identified a number of genes differentially upregulated in S100A4/Mts1 versus control mice. One of these, fibulin-5, is a matrix component necessary for normal elastin fiber assembly. Fibulin-5 was localized to pulmonary arteries and associated with thickened elastic laminae. This feature could underlie attenuation of pulmonary vascular changes in response to elevated pressure, as well as impaired reversibility.

The extracellular superoxide dismutase (ecSOD) plays an important role in atherosclerosis and endothelial function by modulating levels of the superoxide anion (O2*-) in the extracellular space. Although heparan sulfate proteoglycan is an important ligand for ecSOD, little is known about other biological binding partners of ecSOD. The goal of this study was to identify novel proteins that interact with ecSOD. A yeast two-hybrid screening of a human aorta cDNA library using ecSOD as bait identified fibulin-5 as a predominant binding protein for ecSOD. Further analysis showed that the binding domain of ecSOD within fibulin-5 mapped to its C-terminal domain. In vitro pulldown assays and coimmunoprecipitation analysis further confirmed that ecSOD interacts with fibulin-5 in vitro and in vivo. Studies using fibulin-5-/- mice indicated that fibulin-5 is required for binding of ecSOD to vascular tissue. Importantly, the decrease in tissue-bound ecSOD levels in aortas from fibulin-5-/- mice was associated with an increase in vascular O2*- levels. Furthermore, immunohistochemical analysis using ApoE-/- mice suggested a codistribution of ecSOD and fibulin-5 in atherosclerotic vessels. In summary, we provide in this study the first evidence that the ecSOD-fibulin-5 interaction is required for ecSOD binding to vascular tissues, thereby regulating vascular O2*- levels. This interaction may represent a novel mechanism for controlling vascular redox state in the extracellular space in various cardiovascular diseases such as atherosclerosis and hypertension in which oxidative stress is increased.

Extracellular elastic fibres provide mechanical elasticity to tissues and contribute towards the processes of organ remodelling by affecting cell-cell signalling. The formation of elastic fibres requires the assembly and crosslinking of tropoelastin monomers, and organization of the resulting insoluble elastin matrix into functional fibres. The molecules and mechanisms involved in this process are unknown. Fibulin-5 (also known as EVEC/DANCE) is an extracellular matrix protein abundantly expressed in great vessels and cardiac valves during embryogenesis, and in many adult tissues including the aorta, lung, uterus and skin, all of which contain abundant elastic fibres. Here we show that fibulin-5 is a calcium-dependent, elastin-binding protein that localizes to the surface of elastic fibres in vivo. fibulin-5-/- mice develop marked elastinopathy owing to the disorganization of elastic fibres, with resulting loose skin, vascular abnormalities and emphysematous lung. This phenotype, which resembles the cutis laxa syndrome in humans, reveals a critical function for fibulin-5 as a scaffold protein that organizes and links elastic fibres to cells. This function may be mediated by the RGD motif in fibulin-5, which binds to cell surface integrins, and the Ca2+-binding epidermal growth factor (EGF) repeats, which bind elastin.