Beta3-null Mice Research Articles

Absence of beta3 integrin accelerates early skeletal repair

Integrins are heterodimeric transmembrane proteins that mediate cell-matrix interactions and modulate cell behavior. Beta3 subunit is a component of alphaIIbeta3 and alphaVbeta3 integrins. In this study, we first determined that beta3 transcripts are expressed by cells within fracture calluses at 7 and 10 days after injury in a mouse model. We then analyzed fracture healing in mice deficient of beta3 integrin with molecular, histomorphometric, and biomechanical techniques. We found that lack of beta3 integrin results in an extended bleeding time and leads to more bone formation and accelerated cartilage maturation at 7 days after injury. However, beta3 deficiency does not appear to affect later fracture healing. At days 14 and 21, histological appearance or biomechanical properties of fracture calluses are similar between wild type and mutant mice. We also found that altered fracture healing in beta3-null mice is not associated with accelerated angiogenesis, because no significant difference of length density and surface density of blood vessels in fracture limbs was detected at 3 days after injury between wild type and beta3-null mice. In conclusion, our findings demonstrate that beta3 integrin plays an important role during early fracture healing. Further research is required to determine the underlying mechanisms.

Developmental expression analysis of the mouse and chick orthologues of IRF6: The gene mutated in Van der Woude syndrome

Development of the lip and palate involves a complex series of events that are frequently disturbed resulting in the congenital anomalies cleft lip and cleft palate. Van der Woude syndrome (VWS) is an autosomal dominant disorder that is characterised by cleft lip, cleft palate, lower lip pits, and hypodontia. VWS arises as the result of mutations in the gene encoding interferon regulatory factor 6 (IRF6). To provide insights into the role of IRF6 during embryogenesis, we have analysed the expression of this molecule during mouse and chick facial development. Irf6 was expressed in the ectoderm covering the facial processes during their fusion to form the upper lip and primary palate in both mouse and chick. However, while Irf6 was expressed in the medial edge epithelia of the developing secondary palate of the mouse, which fuses as in man, Irf6 was not expressed in the medial edge epithelia of the naturally cleft chick secondary palate. Similarly, Irf6 was found to be down-regulated in the medial edge epithelia of transforming growth factor beta3-null mice, which also exhibit cleft palate. Together, these results support a role for IRF6 during the fusion events that occur during development of the lip and palate.

Beta3-integrin regulates vascular endothelial growth factor-A-dependent permeability.

Beta3-integrin deficiency has been implicated in increasing levels of Flk-1 expression on endothelial cells and enhancing vascular endothelial growth factor (VEGF)-induced angiogenesis. We determined the role of beta3-integrin in mediating VEGF-A-induced blood vessel permeability through Flk-1. Using the Miles assay, we demonstrated that VEGF-A-induced plasma leakage was enhanced in beta3-null mice when compared with wild-type controls. This was not caused by any changes in blood vessel structure (as detected by light or electron microscopy) or by changes in endothelial cell-cell adhesion proteins (as determined by Western blot analysis, flow cytometry, and immunofluorescence). Circulating levels of VEGF, baseline blood vessel leakage, and leakage in response to an acute inflammatory stimulus were identical in wild-type and beta3-null mice. However, VEGF-A-induced leakage was abolished in beta3-null mice by the inhibition of Flk-1, indicating that the elevated levels of Flk-1 on beta3-null endothelial cells enhance VEGF-A-induced permeability. beta3-integrin-deficiency increases the sensitivity of endothelial cells to VEGF-A by elevating Flk-1 expression and, as a consequence, enhances VEGF-A-mediated permeability.

Modified Cardiovascular L-type Channels in Mice Lacking the Voltage-dependent Ca2+ Channel β3 Subunit

The beta subunits of voltage-dependent calcium channels are known to modify calcium channel currents through pore-forming alpha1 subunits. Of the four beta subunits reported to date, the beta3 subunit is highly expressed in smooth muscle cells and is thought to consist of L-type calcium channels. To determine the role of the beta3 subunit in the voltage-dependent calcium channels of the cardiovascular system in situ, we performed a series of experiments in beta3-null mice. Western blot analysis indicated a significant reduction in expression of the alpha1 subunit in the plasma membrane of beta3-null mice. Dihydropyridine binding experiments also revealed a significant decrease in the calcium channel population in the aorta. Electrophysiological analyses indicated a 30% reduction in Ca2+ channel current density, a slower inactivation rate, and a decreased dihydropyridine-sensitive current in beta3-null mice. The reductions in the peak current density and inactivation rate were reproduced in vitro by co-expression of the calcium channel subunits in Chinese hamster ovary cells. Despite the reduced channel population, beta3-null mice showed normal blood pressure, whereas a significant reduction in dihydropyridine responsiveness was observed. A high salt diet significantly elevated blood pressure only in the beta3-null mice and resulted in hypertrophic changes in the aortic smooth muscle layer and cardiac enlargement. In conclusion, this study demonstrates the involvement and importance of the beta3 subunit of voltage-dependent calcium channels in the cardiovascular system and in regulating channel populations and channel properties in vascular smooth muscle cells.

Mice lacking beta3 integrins are osteosclerotic because of dysfunctional osteoclasts.

Osteoclasts express the alphavbeta3 integrin, an adhesion receptor that has been implicated in bone resorption and that is therefore a potential therapeutic target. To assess the role of this heterodimer in skeletal development in vivo, we engineered mice in which the gene for the beta3 integrin subunit was deleted. Bone marrow macrophages derived from these mutants differentiate in vitro into numerous osteoclasts, thus establishing that alphavbeta3 is not necessary for osteoclast recruitment. Furthermore, the closely related integrin, alphavbeta5, does not substitute for alphavbeta3 during cytokine stimulation or authentic osteoclastogenesis. beta3 knockout mice, but not their heterozygous littermates, develop histologically and radiographically evident osteosclerosis with age. Despite their increased bone mass, beta3-null mice contain 3.5-fold more osteoclasts than do heterozygotes. These mutant osteoclasts are, however, dysfunctional, as evidenced by their reduced ability to resorb whale dentin in vitro and the significant hypocalcemia seen in the knockout mice. The resorptive defect in beta3-deficient osteoclasts may reflect absence of matrix-derived intracellular signals, since their cytoskeleton is distinctly abnormal and they fail to spread in vitro, to form actin rings ex vivo, or to form normal ruffled membranes in vivo. Thus, although it is not required for osteoclastogenesis, the integrin alphavbeta3 is essential for normal osteoclast function.

Conserved smooth muscle contractility and blood pressure increase in response to high-salt diet in mice lacking the beta3 subunit of the voltage-dependent calcium channel.

Voltage-dependent calcium channels are crucially important for calcium influx and the following smooth muscle contraction. Beta subunits of these channels are known to modify calcium currents through pore-forming alpha subunits. Among the four reported independent beta subunits, the beta3 subunit is expressed in smooth muscle cells and thought to compose L-type calcium channels in the tissue. To determine the role of the beta3 subunit in the cardiovascular system, we have analyzed beta3-null mice. Electrophysiological examinations proved the existence of dihydropyridine (DHP)-sensitive. L-type calcium channels in the smooth muscle cells. Beta3-null mice show no apparent changes in smooth muscle contraction and sensitivity to DHP, and normal blood pressure when they are raised on a normal diet, but the 13 subunit deficient mice show elevated blood pressure in response to a high-salt diet, with significant reductions in plasma catecholamine concentrations. Our finding strongly suggests a close relationship between voltage-dependent channels and high blood pressure.

Beta3-integrin-deficient mice are a model for Glanzmann thrombasthenia showing placental defects and reduced survival.

beta3 integrins have been implicated in a wide variety of functions, including platelet aggregation and thrombosis (alphaIIbbeta3) and implantation, placentation, angiogenesis, bone remodeling, and tumor progression (alphavbeta3). The human bleeding disorder Glanzmann thrombasthenia (GT) can result from defects in the genes for either the alphaIIb or the beta3 subunit. In order to develop a mouse model of this disease and to further studies of hemostasis, thrombosis, and other suggested roles of beta3 integrins, we have generated a strain of beta3-null mice. The mice are viable and fertile, and show all the cardinal features of GT (defects in platelet aggregation and clot retraction, prolonged bleeding times, and cutaneous and gastrointestinal bleeding). Implantation appears to be unaffected, but placental defects do occur and lead to fetal mortality. Postnatal hemorrhage leads to anemia and reduced survival. These mice will allow analyses of the other suggested functions of beta3 integrins and we report that postnatal neovascularization of the retina appears to be beta3-integrin-independent, contrary to expectations from inhibition experiments.