Masahito Horiguchi

Latent TGF-β-binding proteins

The LTBPs (or latent transforming growth factor β 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 TGFβ by covalently binding the TGFβ propeptide (LAP) via disulfide bonds in the endoplasmic reticulum. LAP in turn is cleaved from the mature TGFβ precursor in the trans-golgi network but LAP and TGFβ remain strongly bound through non-covalent interactions. LAP, TGFβ, and LTBP together form the large latent complex (LLC). LTBPs were originally thought to primarily play a role in maintaining TGFβ latency and targeting the latent growth factor to the extracellular matrix (ECM), but it has also been shown that LTBP-1 participates in TGFβ 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 TGFβ regulation, TGFβ-independent activities have recently been identified for LTBP-2 and LTBP-4 in stabilizing microfibril bundles and regulating elastic fiber assembly.

Specificity of latent TGF-β binding protein (LTBP) incorporation into matrix: Role of fibrillins and fibronectin†

Fibrillin microfibrils are extracellular matrix structures with essential functions in the development and the organization of tissues including blood vessels, bone, limbs and the eye. Fibrillin‐1 and fibrillin‐2 form the core of fibrillin microfibrils, to which multiple proteins associate to form a highly organized structure. Defining the components of this structure and their interactions is crucial to understand the pathobiology of microfibrillopathies associated with mutations in fibrillins and in microfibril‐associated molecules. In this study, we have analyzed both in vitro and in vivo the role of fibrillin microfibrils in the matrix deposition of latent TGF‐β binding protein 1 (LTBP‐1), ‐3 and ‐4; the three LTBPs that form a complex with TGF‐β. In Fbn1−/− ascending aortas and lungs, LTBP‐3 and LTBP‐4 are not incorporated into a matrix lacking fibrillin‐1 microfibrils, whereas LTBP‐1 is still deposited. In addition, in cultures of Fbn1−/− smooth muscle cells or lung fibroblasts, LTBP‐3 and LTBP‐4 are not incorporated into a matrix lacking fibrillin‐1 microfibrils, whereas LTBP‐1 is still deposited. Fibrillin‐2 is not involved in the deposition of LTBP‐1 in Fbn1−/− extracellular matrix as cells deficient for both fibrillin‐1 and fibrillin‐2 still incorporate LTBP‐1 in their matrix. However, blocking the formation of the fibronectin network in Fbn1−/− cells abrogates the deposition of LTBP‐1. Together, these data indicate that LTBP‐3 and LTBP‐4 association with the matrix depends on fibrillin‐1 microfibrils, whereas LTBP‐1 association depends on a fibronectin network. J. Cell. Physiol. 227: 3828–3836, 2012.

Latent TGF-β binding protein 4 promotes elastic fiber assembly by interacting with fibulin-5

Elastic fiber assembly requires deposition of elastin monomers onto microfibrils, the mechanism of which is incompletely understood. Here we show that latent TGF-β binding protein 4 (LTBP-4) potentiates formation of elastic fibers through interacting with fibulin-5, a tropoelastin-binding protein necessary for elastogenesis. Decreased expression of LTBP-4 in human dermal fibroblast cells by siRNA treatment abolished the linear deposition of fibulin-5 and tropoelastin on microfibrils. It is notable that the addition of recombinant LTBP-4 to cell culture medium promoted elastin deposition on microfibrils without changing the expression of elastic fiber components. This elastogenic property of LTBP-4 is independent of bound TGF-β because TGF-β–free recombinant LTBP-4 was as potent an elastogenic inducer as TGF-β–bound recombinant LTBP-4. Without LTBP-4, fibulin-5 and tropoelastin deposition was discontinuous and punctate in vitro and in vivo. These data suggest a unique function for LTBP-4 during elastic fibrogenesis, making it a potential therapeutic target for elastic fiber regeneration.

Latent TGF-β binding protein-2 is essential for the development of ciliary zonule microfibrils

Latent TGF-β-binding protein-2 (LTBP-2) is an extracellular matrix protein associated with microfibrils. Homozygous mutations in LTBP2 have been found in humans with genetic eye diseases such as congenital glaucoma and microspherophakia, indicating a critical role of the protein in eye development, although the function of LTBP-2 in vivo has not been well understood. In this study, we explore the in vivo function of LTBP-2 by generating Ltbp2(-/-) mice. Ltbp2(-/-) mice survived to adulthood but developed lens luxation caused by compromised ciliary zonule formation without a typical phenotype related to glaucoma, suggesting that LTBP-2 deficiency primarily causes lens dislocation but not glaucoma. The suppression of LTBP2 expression in cultured human ciliary epithelial cells by siRNA disrupted the formation of the microfibril meshwork by the cells. Supplementation of recombinant LTBP-2 in culture medium not only rescued the microfibril meshwork formation in LTBP2-suppressed ciliary epithelial cells but also restored unfragmented and bundled ciliary zonules in Ltbp2(-/-) mouse eyes under organ culture. Although several reported human mutant LTBP-2 proteins retain normal domain structure and keep the fibrillin-1-binding site intact, none of these mutant proteins were secreted from their producing cells, suggesting secretion arrest occurred to the LTBP-2 mutants owing to conformational alteration. The findings of this study suggest that LTBP-2 is an essential component for the formation of microfibril bundles in ciliary zonules.

Production of Gastrointestinal Tumors in Mice by Modulating Latent TGF-β1 Activation

TGF-β and its signaling pathways are important mediators in the suppression of cancers of the gastrointestinal tract. TGF-β is released from cells in a latent complex consisting of TGF-β, the TGF-β propeptide [latency associated protein (LAP)], and a latent TGF-β binding protein (LTBP). We previously generated mice in which the LTBP-binding cysteine residues in LAP TGF-β1 were mutated to serine precluding covalent interactions with LTBP. These Tgfb1(C33S/C33S) mice develop multiorgan inflammation and tumors consistent with reduced TGF-β1 activity. To test whether further reduction in active TGF-β levels would yield additional tumors and a phenotype more similar to Tgfb1(-/-) mice, we generated mice that express TGF-β1(C33S) and are deficient in either integrin β8 or TSP-1, known activators of latent TGF-β1. In addition, we generated mice that have one mutant allele and one null allele at the Tgfb1 locus, reasoning that these mice should synthesize half the total amount of TGF-β1 as Tgfb1(C33S/C33S) mice, and the amount of active TGF-β1 would be correspondingly decreased compared with Tgfb1(C33S/C33S) mice. These compound-mutant mice displayed more severe inflammation and higher tumor numbers than the parental Tgfb1(C33S/C33S) animals. The level of active TGF-β1 in compound mutant mice seemed to be decreased compared with Tgfb1(C33S/C33S) mice as determined from analyses of surrogate markers of active TGF-β, such as P-Smad2, C-Myc, KI-67, and markers of cell-cycle traverse. We conclude that these mutant mice provide a useful system for modulating TGF-β levels in a manner that determines tumor number and inflammation within the gastrointestinal tract.

Genetic Suppression of Inflammation Blocks the Tumor-Promoting Effects of TGF-β in Gastric Tissue

The contributions of TGF-β signaling to cancer are complex but involve the inflammatory microenvironment as well as cancer cells themselves. In mice encoding a TGF-β mutant that precludes its binding to the latent TGF-β 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-β1. By genetically eliminating activators of latent TGF-β1, we further lowered the amount of TGF-β, which enhanced tumor frequency and multiorgan inflammation. This model system was used to further investigate the relative contribution of TGF-β1 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 with wild-type animals, whereas tissue from Tgfb1(-/C33S);Rag2(-/-) mice had normal P-Smad3 levels. Inhibiting the inflammatory response normalized levels of interleukin (IL)-1β and IL-6 and reduced tumor cell proliferation. In addition, 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-β1 may be directly or indirectly affected by inflammatory processes, which accompany loss of TGF-β1, rather than a direct effect of loss of the cytokine.