Faculty Bibliography

The LTBPs (or latent transforming growth factor beta binding proteins) are important components of the extracellular matrix (ECM) that interact with fibrillin microfibrils and have a number of different roles in microfibril biology. There are four LTBPs isoforms in the human genome (LTBP-1, -2, -3, and -4), all of which appear to associate with fibrillin and the biology of each isoform is reviewed here. The LTBPs were first identified as forming latent complexes with TGFbeta by covalently binding the TGFbeta propeptide (LAP) via disulfide bonds in the endoplasmic reticulum. LAP in turn is cleaved from the mature TGFbeta precursor in the trans-golgi network but LAP and TGFbeta remain strongly bound through non-covalent interactions. LAP, TGFbeta, and LTBP together form the large latent complex (LLC). LTBPs were originally thought to primarily play a role in maintaining TGFbeta latency and targeting the latent growth factor to the extracellular matrix (ECM), but it has also been shown that LTBP-1 participates in TGFbeta activation by integrins and may also regulate activation by proteases and other factors. LTBP-3 appears to have a role in skeletal formation including tooth development. As well as having important functions in TGFbeta regulation, TGFbeta-independent activities have recently been identified for LTBP-2 and LTBP-4 in stabilizing microfibril bundles and regulating elastic fiber assembly.

Mutations in the gene for the latent transforming growth factor beta binding protein 4 (LTBP4) cause autosomal recessive cutis laxa type 1C. To understand the molecular disease mechanisms of this disease, we investigated the impact of LTBP4 loss on transforming growth factor beta (TGFbeta) signaling. Despite elevated extracellular TGFbeta activity, downstream signaling molecules of the TGFbeta pathway, including pSMAD2 and pERK, were down-regulated in LTBP4 mutant human dermal fibroblasts. In addition, TGFbeta receptors 1 and 2 (TGFBR1 and TGFBR2) were reduced at the protein but not at the ribonucleic acid level. Treatment with exogenous TGFbeta1 led to an initially rapid increase in SMAD2 phosphorylation followed by a sustained depression of phosphorylation and receptor abundance. In mutant cells TGFBR1 was co-localized with lysosomes. Treatment with a TGFBR1 kinase inhibitor, endocytosis inhibitors or a lysosome inhibitor, normalized the levels of TGFBR1 and TGFBR2. Co-immunoprecipitation demonstrated a molecular interaction between LTBP4 and TGFBR2. Knockdown of LTBP4 reduced TGFbeta receptor abundance and signaling in normal cells and supplementation of recombinant LTBP4 enhanced these measures in mutant cells. In a mouse model of Ltbp4 deficiency, reduced TGFbeta signaling and receptor levels were normalized upon TGFBR1 kinase inhibitor treatment. Our results show that LTBP4 interacts with TGFBR2 and stabilizes TGFbeta receptors by preventing their endocytosis and lysosomal degradation in a ligand-dependent and receptor kinase activity-dependent manner. These findings identify LTBP4 as a key molecule required for the stability of the TGFbeta receptor complex, and a new mechanism by which the extracellular matrix regulates cytokine receptor signaling.

Inherited dental malformations constitute a clinically and genetically heterogeneous group of disorders. Here, we report on four families, three of them consanguineous, with an identical phenotype, characterized by significant short stature with brachyolmia and hypoplastic amelogenesis imperfecta (AI) with almost absent enamel. This phenotype was first described in 1996 by Verloes et al. as an autosomal recessive form of brachyolmia associated with AI. Whole exome sequencing resulted in the identification of recessive hypomorphic mutations including deletion, nonsense and splice mutations, in the LTBP3 gene, which is involved in the TGF-beta signaling pathway. We further investigated gene expression during mouse development and tooth formation. Differentiated ameloblasts synthesizing enamel matrix proteins and odontoblasts expressed the gene. Study of an available knockout mouse model showed that the mutant mice displayed very thin to absent enamel in both incisors and molars, hereby recapitulating the amelogenesis imperfecta phenotype in the human disorder.

Mice deficient in Latent TGFbeta Binding Protein 4 (Ltbp4) display a defect in lung septation and elastogenesis. The lung septation defect is normalized by genetically decreasing TGFbeta2 levels. However, the elastic fiber assembly is not improved in Tgfb2(-/-) ;Ltbp4S(-/-) compared to Ltbp4S(-/-) lungs. We found that decreased levels of TGFbeta1 or TGFbeta3 did not improve lung septation indicating that the TGFbeta isoform elevated in Ltbp4S(-/-) lungs is TGFbeta2. Expression of a form of Ltbp4 that could not bind latent TGFbeta did not affect lung phenotype indicating that normal lung development does not require the formation of LTBP4-latent TGFbeta complexes. Therefore, the change in TGFbeta-level in the lungs is not directly related to Ltbp4 deficiency but probably is a consequence of changes in the extracellular matrix. Interestingly, combination of the Ltbp4S(-/-) mutation with a fibulin-5 null mutant in Fbln5(-/-) ;Ltbp4S(-/-) mice improves the lung septation compared to Ltbp4S(-/-) lungs. Large globular elastin aggregates characteristic for Ltbp4S(-/-) lungs do not form in Fbln5(-/-) ;Ltbp4S(-/-) lungs and EM studies showed that elastic fibers in Fbln5(-/-) ;Ltbp4S(-/-) lungs resemble those found in Fbln5(-/-) mice. These results are consistent with a role for TGFbeta2 in lung septation and for Ltbp4 in regulating fibulin-5 dependent elastic fiber assembly. J. Cell. Physiol. 229: 226-236, 2014. (c) 2014 Wiley Periodicals, Inc.

Over the past two decades, the secreted protein sonic hedgehog (SHH) has emerged as a critical morphogen during embryonic lung development, regulating the interaction between epithelial and mesenchymal cell populations in both the airway and alveolar compartments. There is increasing evidence that the SHH pathway is active in adult lung diseases such as pulmonary fibrosis, asthma, chronic obstructive pulmonary disease (COPD) and lung cancer, which raises two questions: (1) what role does SHH signaling play in these diseases? (2) Is it a primary driver of the disease, or a response (perhaps beneficial) to the primary disturbance? In this review we aim to fill the gap between the well-studied period of embryonic lung development and the adult diseased lung by reviewing the HH pathway during the postnatal period, and in adult uninjured and injured lungs. We elucidate the similarities and differences in the epithelial-mesenchymal interplay during the fibrosis response to injury in lung compared to other organs, and present a critical appraisal of tools and agents available to evaluate HH signaling.

Anchoring cell junctions are integral in maintaining electro-mechanical coupling of ventricular working cardiomyocytes; however, their role in cardiomyocytes of the cardiac conduction system (CCS) remains less clear. Recent studies in genetic mouse models and humans highlight the appearance of these cell junctions alongside gap junctions in the CCS and also show that defects in these structures and their components are associated with conduction impairments in the CCS. Here we outline current evidence supporting an integral relationship between anchoring and gap junctions in the CCS. Specifically we focus on (1) molecular and ultrastructural evidence for cell-cell junctions in specialized cardiomyocytes of the CCS, (2) genetic mouse models specifically targeting cell-cell junction components in the heart which exhibit CCS conduction defects and (3) human clinical studies from patients with cell-cell junction-based diseases that exhibit CCS electrophysiological defects.

The contributions of TGF-beta signaling to cancer are complex but involve the inflammatory microenvironment as well as cancer cells themselves. In mice encoding a TGF-beta mutant that precludes its binding to the latent TGF-beta binding protein (Tgfb1-/C33S), we observed multiorgan inflammation and an elevated incidence of various types of gastrointestinal solid tumors due to impaired conversion of latent to active TGF-beta1. By genetically eliminating activators of latent TGF-beta, we further lowered the amount of TGF-beta, which enhanced tumor frequency and multiorgan inflammation. This model system was used to further investigate the relative contribution of TGF-beta1 to lymphocyte-mediated inflammation in gastrointestinal tumorigenesis. Toward this end, we generated Tgfb1-/C33S;Rag2-/- mice that lacked adaptive immune function, which eliminated tumor production. Analysis of tissue from Tgfb1-/C33S mice indicated decreased levels of P-Smad3 compared to wild type animals, whereas tissue from Tgfb1-/C33S;Rag2-/- mice had normal P-Smad3 levels. Inhibiting the inflammatory response normalized levels of IL-1beta and IL-6 and reduced tumor cell proliferation. Additionally, Tgfb1-/C33S;Rag2-/- mice exhibited reduced paracrine signaling in the epithelia, mediated by hepatocyte growth factor produced by gastric stroma. Together, our results indicate that many of the responses of the gastric tissue associated with decreased TGF-beta1 may be directly or indirectly affected by inflammatory processes, which accompany loss of TGF-beta1, rather than a direct effect of loss of the cytokine.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) termed a 'disease of the desmosome' is an inherited cardiomyopathy that recently underwent reclassification owing to the identification of left-dominant and biventricular disease forms. Homozygous loss-of-function mutations in the desmosomal component, desmoplakin, are found in patients exhibiting a biventricular form of ARVC; however, no models recapitulate the postnatal hallmarks of the disease as seen in these patients. To gain insights into the homozygous loss-of-function effects of desmoplakin in the heart, we generated cardiomyocyte-specific desmoplakin-deficient mice (DSP-cKO) using ventricular myosin light chain-2-Cre mice. Homozygous DSP-cKO mice are viable but display early ultrastructural defects in desmosomal integrity leading to a cardiomyopathy reminiscent of a biventricular form of ARVC, which includes cell death and fibro-fatty replacement within the ventricle leading to biventricular dysfunction, failure and premature death. DSP-cKO mice also exhibited ventricular arrhythmias that are exacerbated with exercise and catecholamine stimulation. Furthermore, DSP-cKO hearts exhibited right ventricular conduction defects associated with loss of connexin 40 expression and electrical wavefront propagation defects associated with loss of connexin 43 expression. Dose-dependent assessment of the effects of loss of desmoplakin in neonatal ventricular cardiomyocytes revealed primary loss of connexin 43 levels, phosphorylation and function independent of the molecular dissociation of the mechanical junction complex and fibro-fatty manifestation associated with ARVC, suggesting a role for desmoplakin as a primary stabilizer of connexin integrity. In summary, we provide evidence for a novel mouse model, which is reminiscent of the postnatal onset of ARVC while highlighting mechanisms underlying a biventricular form of human ARVC.