Faculty Bibliography

The multifunctional cytokine transforming growth factor (TGF) beta1 is secreted in a latent complex with its processed propeptide (latency-associated peptide [LAP]). TGFbeta1 must be functionally released from this complex before it can engage TGFbeta receptors. One mechanism of latent TGFbeta1 activation involves interaction of the integrins alphavbeta6 and alphavbeta8 with an RGD sequence in LAP; other putative latent TGFbeta1 activators include thrombospondin-1, oxidants, and various proteases. To assess the contribution of RGD-binding integrins to TGFbeta1 activation in vivo, we created a mutation in Tgfb1 encoding a nonfunctional variant of the RGD sequence (RGE). Mice with this mutation (Tgfb1(RGE/RGE)) display the major features of Tgfb1(-/-) mice (vasculogenesis defects, multiorgan inflammation, and lack of Langerhans cells) despite production of normal levels of latent TGFbeta1. These findings indicate that RGD-binding integrins are requisite latent TGFbeta1 activators during development and in the immune system

Fibrosis is a common pathological feature observed in muscles of patients with Duchenne muscular dystrophy (DMD). Biglycan and decorin are small chondroitin/dermatan sulfate proteoglycans in the muscle extracellular matrix (ECM) that belong to the family of structurally related proteoglycans called small leucine-rich repeat proteins. Decorin is considered an anti-fibrotic agent, preventing the process by blocking TGF-beta activity. There is no information about their expression in DMD patients. We found an increased amount of both proteoglycans in the ECM of skeletal muscle biopsies obtained from DMD patients. Both biglycan and decorin were augmented in the perimysium of muscle tissue, but only decorin increased in the endomysium as seen by immunohistochemical analyses. Fibroblasts were isolated from explants obtained from muscle of DMD patients and the incorporation of radioactive sulfate showed an increased synthesis of both decorin and biglycan in cultured fibroblasts compared to controls. The size of decorin and biglycan synthesized by DMD and control fibroblasts seems to be similar in size and anion charge. These findings show that decorin and biglycan are increased in DMD skeletal muscle and suggest that fibroblasts would be, at least, one source for these proteoglycans likely playing a role in the muscle response to dystrophic cell damage.

The membranous bones of the mammalian skull vault arise from discrete condensations of neural crest- and mesodermally-derived cells. Recently, a number of homeodomain transcription factors have been identified as critical regulators of this process. Here, we show that the homeoprotein engrailed 1 (EN1) is expressed during embryonic and perinatal craniofacial bone development, where it localizes to the skeletogenic mesenchyme, and, subsequently, to calvarial osteoblasts and osteoprogenitors. Mice lacking En1 exhibit generalized calvarial bone hypoplasia and persistent widening of the sutural joints. A reduction in calvarial membranous bone deposition and mineralization (osteopenia) is coupled to enhanced osteolytic resorption in En1 mutants. Consistent with these observations, expression of established osteoblast differentiation markers reveals that En1 function is required for both early and late phases of calvarial osteogenesis. Further analysis shows that EN1 regulates FGF signaling in calvarial osteoblasts. Moreover, EN1 indirectly influences calvarial osteoclast recruitment and bone resorption by regulating the expression of receptor activator of NFkappaB ligand (RANKL) in osteoblasts. Thus, during intramembranous bone formation, EN1 acts both cell autonomously and non-cell autonomously. In summary, this study identifies EN1 as a novel modulator of calvarial osteoblast differentiation and proliferation, processes that must be exquisitely balanced to ensure proper skull vault formation

Latent transforming growth factor (TGF)-beta binding proteins (LTBPs) modulate the secretion and activation of latent TGF-beta. To explore LTBP function in vivo, we created an Ltbp-3(-/-) mouse that has developmental emphysema with decreased septation in terminal alveoli. Differences in distal airspace enlargement were obvious at day 6 after birth. Secondary septation was inhibited, so by days 21 to 28 the mean linear intercept was approximately twofold greater in mutant versus control lungs. There were no differences in lung collagen and elastin, visualized by immunohistochemistry, or in myofibroblast numbers, determined by alpha-smooth muscle actin-positive cells, between mutant or wild-type lungs as the animals aged, other than differences associated with altered lung structure in mutant animals. However, from day 10 there was twice the number of alveolar type II cells in mutant alveoli compared to controls. At days 6 and 10, a transient enhancement in cell proliferation in the mutant lungs was observed by both 5-bromo-2'-deoxy-uridine and proliferating cell nuclear antigen labeling, accompanied by enhanced numbers of terminal dUTP nick-end labeling-positive cells at days 4, 6, and 10. Finally, there was a transient decrease in TGF-beta signaling at days 4 to 6 in Ltbp-3(-/-) lungs. These results indicate that in the absence of Ltbp-3, a temporary decrease in TGF-beta signaling in the lungs at days 4 to 6 alters cell proliferation, correlating with inhibition of septation and developmental emphysema

LTBPs are extracellular matrix proteins resembling fibrillins. LTBP-1, 3, and 4 covalently bind latent TGF-beta and modulate tissue levels of this potent cytokine through regulation of its secretion, localization, and/or activation. To address LTBP function in vivo, we generated Ltbp-3 null mice. Ltbp-3-/- animals developed craniofacial abnormalities due to early ossification of the skull base synchondroses and displayed reduced body size. In addition, histological examination of Ltbp-3-/- skeletons revealed an increase in bone mass. The osteoblast numbers and mineral apposition rates were decreased in Ltbp-3-/- mice, whereas the osteoclast numbers were similar in null and wild type mice. Histological examination revealed persistence of cartilage remnants in Ltbp-3-/- trabecular bone. Taken together, these results indicate that the Ltbp-3-/- high bone mass phenotype was due to a defect in bone resorption. We hypothesize that lack of Ltbp-3 results in decreased levels of TGF-beta in bone and cartilage, which leads to compromised osteoclast function and decreased bone turnover

Latent TGF-beta binding proteins are multidomain proteins with a common, highly repetitive structural organization and partially overlapping expression patterns. Latent TGF-beta binding protein-1, -3 and -4 bind latent TGF-beta. TGF-betas are normally secreted as latent complexes, consisting of the mature TGF-beta dimer non-covalently bound to its processed propeptide dimer plus a latent TGF-beta binding protein. The latent TGF-beta binding protein is covalently bound to the propeptide. These binding proteins may perform at least two functions: structural, as components of the matrix, and regulatory, as modulators of TGF-beta availability

The transforming growth factor (TGF)-betas are essential in pre- and postnatal development, differentiation and morphogenesis of higher organisms. Quantitation of the levels of TGF-beta synthesis, secretion, and activation are crucial for grasping the mechanisms that control these events and ultimately control TGF-beta action. Rather than presenting a single method, we describe several methods for measuring active TGF-beta in different experimental situations. This is possible as a result of advances in transgenic mice technology that allow in vivo TGF-beta measurements in addition to the more established in vitro approaches