Faculty Bibliography

Sonic Hedgehog (Shh) signals from epithelium to mesenchyme during embryonic lung development, but the roles of Hedgehog (Hh) signaling in postnatal lung development and adult lung are not known. Using Gli1nlacZ reporter mice to identify cells with active Hh signaling, we found that Gli1nlacZ-positive mesenchymal cells are densely and diffusely present up to 2 weeks after birth and decline in number thereafter. In adult mice, Gli1nlacZ-positive cells are present around large airways and vessels and are sparse in alveolar septa. Hh-stimulated cells are mostly fibroblasts; only 10% of Gli1nlacZ-positive cells are smooth muscle cells, and most smooth muscle cells do not have activation of Hh signaling. After bleomycin injury there are abundant Gli1nlacZ-positive mesenchymal cells in fibrotic lesions and increased numbers of Gli1nlacZ-positive cells in preserved alveolar septa. Inhibition of Hh signaling with an antibody against all Hedgehog isoforms does not reduce bleomycin-induced fibrosis, but adenovirus-mediated over-expression of Shh increases collagen production in this model. Inhibition of Hh signaling during early postnatal lung development causes airspace enlargement without diminished alveolar septation. Reduction of Hh signaling in the later stages of postnatal lung development may be required for normal thinning and maturation of alveolar septa.

The growth factor TGF-beta is secreted in a latent complex consisting of three proteins: TGF-beta, an inhibitor (latency-associated protein, LAP, which is derived from the TGF-beta propeptide) and an ECM-binding protein (one of the latent TGF-beta binding proteins, or LTBPs). LTBPs interact with fibrillins and other ECM components and thus function to localize latent TGF-beta in the ECM. LAP contains an integrin-binding site (RGD), and several RGD-binding integrins are able to activate latent TGF-beta through binding this site. Mutant mice defective in integrin-mediated activators, and humans and mice with fibrillin gene mutations, show the critical role of ECM and integrins in regulating TGF-beta signaling

The arginine-glycine-aspartate (RGD)-binding integrins alphavbeta6 and alphavbeta8 activate latent TGFbeta1 and TGFbeta3 in vivo, but it is uncertain whether other RGD-binding integrins such as integrins alphavbeta5 and alphavbeta3 activate these TGFbeta isoforms. To define the combined role of alphavbeta6- and alphavbeta8-integrin in TGFbeta activation, we analyzed mice lacking function of both integrins by means of gene deletion and/or pharmacologic inhibition. Most Itgb6-/-;Itgb8-/- embryos die at mid-gestation; those that survive develop cleft palate-as observed in Tgfb3-/- mice. Itgb8-/- mice treated with an anti-alphavbeta6-integrin antibody develop severe autoimmunity and lack Langerhans cells-similar to Tgfb1-null mice. These results support a model in which TGFbeta3-mediated palate fusion and TGFbeta1-mediated suppression of autoimmunity and generation of Langerhans cells require integrins alphavbeta6 and alphavbeta8 but not other RGD-binding integrins as TGFbeta activators

Gene deletion experiments have shown that the three TGFbeta isoforms regulate distinct developmental processes. Recent work by our group and others showed that the integrins alphavbeta6 and alphavbeta8 activate latent forms of TGFbeta1 and TGFbeta3. This raises the possibility that TGFbeta1 and TGFbeta3 act redundantly in developmental processes where both isoforms are expressed and activation is by integrins. To investigate this issue, we generated mice with defective integrin-mediated TGFbeta1 activation (Tgfb1(RGE/RGE)) that were also homozygous for a null mutation in the TGFbeta3 gene. Tgfb1(RGE/RGE); Tgfb3(-/-) mice have severely perturbed development of the brain vasculature that is highly similar to that in mice lacking alphavbeta8. Some Tgfb1(RGE/RGE); Tgfb3(+/-) and Tgfb1(RGE/RGE); Tgfb3(+/+) mice have milder, background-dependent versions of the phenotype. In addition, we found that Tgfb3 gene status influences embryonic lethality due to TGFbeta1 deficiency after limited backcrossing to the BALB/c background. Conversely, Tgfb1 gene status modifies the extent of palate fusion in Tgfb3(-/-) mice after limited backcrossing to the ICR background. Our results are consistent with a functional connection between TGFbeta1 and TGFbeta3 during development based on a shared mechanism of activation