Faculty Bibliography

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.

Much of our understanding of gut-microbial interactions has come from mouse models. Intestinal immunity is complex and a combination of host genetics and environmental factors play a significant role in regulating intestinal immunity. Due to this complexity, no mouse model to date gives a complete and accurate representation of human intestinal diseases, such as inflammatory bowel diseases. However, intestinal tissue from patients undergoing bowel resection reflects a condition of severe disease that has failed treatment, hence a more dynamic perspective of varying inflammatory states in IBD could be obtained through the analyses of pinch biopsy material. Here we describe our protocol for analyzing mucosal pinch biopsies collected predominantly during colonoscopies. We have optimized flow cytometry panels to analyze up to 8 cytokines produced by CD4+ and CD8+ cells, as well as for characterizing nuclear proteins and transcription factors such as Ki67 and Foxp3. Furthermore, we have optimized approaches to analyze the production of cytokines, including TGF-beta from direct ex vivo cultures of pinch biopsies and LPMCs isolated from biopsies. These approaches are part of our workflow to try and understand the role of the gut microbiota in complex and dynamic human intestinal diseases.

Latent transforming growth factor-beta binding protein-1 (LTBP-1) is an extracellular protein that is structurally similar to fibrillin and has an important role in controlling transforming growth factor-beta (TGF-beta) signaling by storing the cytokine in the extracellular matrix and by being involved in the conversion of the latent growth factor to its active form. LTBP-1 is found as both short (LTBP-1S) and long (LTBP-1L) forms, which are derived through the use of separate promoters. There is controversy regarding the importance of LTBP-1L, as Ltbp1L knockout mice showed multiple cardiovascular defects but the complete null mice did not. Here, we describe a third line of Ltbp1 knockout mice generated utilizing a conditional knockout strategy that ablated expression of both L and S forms of LTBP-1. These mice show severe developmental cardiovascular abnormalities and die perinatally; thus these animals display a phenotype similar to previously reported Ltbp1L knockout mice. We reinvestigated the other "complete" knockout line and found that these mice express a splice variant of LTBP-1L and, therefore, are not complete Ltbp1 knockouts. Our results clarify the phenotypes of Ltbp1 null mice and re-emphasize the importance of LTBP-1 in vivo.

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.

BACKGROUND: Hepatic fibrosis, which is the excessive accumulation of extracellular matrices (ECMs) produced mainly from activated hepatic stellate cells (HSCs), develops to cirrhosis over several decades. There are no validated biomarkers that can non-invasively monitor excessive production of ECM (i.e., fibrogenesis). Transforming growth factor (TGF)-beta, a key driver of fibrogenesis, is produced as an inactive latent complex, in which active TGF-beta is enveloped by its pro-peptide, the latency-associated protein (LAP). Thus, active TGF-beta must be released from the complex for binding to its receptor and inducing ECM synthesis. We recently reported that during the pathogenesis of liver fibrosis, plasma kallikrein (PLK) activates TGF-beta by cleavage between R(58) and L(59) residues within LAP and that one of its by-products, the N-terminal side LAP degradation products ending at residue R(58) (R(58) LAP-DPs), can be detected mainly around activated HSCs by specific antibodies against R(58) cleavage edges and functions as a footprint of PLK-dependent TGF-beta activation. Here, we describe a sandwich enzyme-linked immunosorbent assay (ELISA) that detects the other by-products, the C-terminal side LAP-DPs starting from residue L(59) (L(59) LAP-DPs). We demonstrated that the L(59) LAP-DPs are a potentially novel blood biomarker reflecting hepatic fibrogenesis. RESULTS: We established a specific sandwich ELISA to quantify L(59) LAP-DPs as low as 2 pM and measured L(59) LAP-DP levels in the culture media of a human activated HSC line, TWNT-4 cells. L(59) LAP-DPs could be detected in their media, and after treatment of TWNT-4 cells with a TGF-beta receptor kinase inhibitor, SB431542, a simultaneous reduction was observed in both L(59) LAP-DP levels in the culture media and the mRNA expression levels of collagen type (I) alpha1. In carbon tetrachloride- and bile duct ligation-induced liver fibrosis models in mice, plasma L(59) LAP-DP levels increased prior to increase of hepatic hydroxyproline (HDP) contents and well correlated with alpha-smooth muscle actin (alphaSMA) expression in liver tissues. At this time, alphaSMA-positive cells as well as R(58) LAP-DPs were seen in their liver tissues. CONCLUSIONS: L(59) LAP-DPs reflect PLK-dependent TGF-beta activation and the increase in alphaSMA-positive activated HSCs in liver injury, thereby serving as a novel blood biomarker for liver fibrogenesis.

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.

The large intestine is the site most commonly affected in inflammatory bowel diseases. However, the mechanism of T cell homing to the large intestine, which contributes to inflammation, had remained unclear. We show here that an orphan G-protein coupled receptor GPR15 controls the specific homing of T cells, particularly FOXP3+ regulatory T cells (Tregs), to the large intestine lamina propria (LILP). GPR15 expression is promoted by gut microbiota and TGF-beta1, but not by retinoic acid. GPR15-deficient mice had fewer Tregs in LILP and were prone to develop more severe inflammation in the large intestine, which was rescued by the transfer of GPR15-sufficient Tregs. Our findings thus indicate that GPR15 is a T cell homing receptor for LILP and that GPR15 plays a key role in maintaining gut immune homeostasis, largely by regulating the influx of Tregs. Our study also demonstrates that adaptive immune responses in the gut are functionally compartmentalized through the differential requirements for T cell homing to the small and large bowel

Kindlin-1 is an integrin tail binding protein that controls integrin activation. Mutations in the FERMT-1 gene, which encodes for Kindlin-1, lead to Kindler syndrome in man, which is characterized by skin blistering, premature skin aging and skin cancer of unknown etiology. Here we show that loss of Kindlin-1 in mouse keratinocytes recapitulates Kindler syndrome and also produces enlarged and hyperactive stem cell compartments, which lead to hyperthickened epidermis, ectopic hair follicle development and increased skin tumor susceptibility. Mechanistically, Kindlin-1 controls keratinocyte adhesion through beta1-class integrins and proliferation and differentiation of cutaneous epithelial stem cells by promoting alphavbeta6 integrin-mediated transforming growth factor-beta (TGF-beta) activation and inhibiting Wnt-beta-catenin signaling through integrin-independent regulation of Wnt ligand expression. Our findings assign Kindlin-1 the previously unknown and essential task of controlling cutaneous epithelial stem cell homeostasis by balancing TGF-beta-mediated growth-inhibitory signals and Wnt-beta-catenin-mediated growth-promoting signals.

Transforming growth factor-betas (TGF-betas) are secreted from cells as latent complexes and the activity of TGF-betas is controlled predominantly through activation of these complexes. Tolerance to the fetal allograft is essential for pregnancy success; TGF-beta1 and TGF-beta2 play important roles in regulating these processes. Pregnancy-specific beta-glycoproteins (PSGs) are present in the maternal circulation at a high concentration throughout pregnancy and have been proposed to have anti-inflammatory functions. We found that recombinant and native PSG1 activate TGF-beta1 and TGF-beta2 in vitro. Consistent with these findings, administration of PSG1 protected mice from dextran sodium sulfate (DSS)-induced colitis, reduced the secretion of pro-inflammatory cytokines, and increased the number of T regulatory cells. The PSG1-mediated protection was greatly inhibited by the coadministration of neutralizing anti-TGF-beta antibody. Our results indicate that proteins secreted by the placenta directly contribute to the generation of active TGF-beta and identify PSG1 as one of the few known biological activators of TGF-beta2.Mucosal Immunology advance online publication, 14 August 2013; doi:10.1038/mi.2013.53.