Faculty Bibliography

Latent transforming growth factor β (TGFβ)-binding proteins (LTBPs) are important for the secretion, activation, and function of mature TGFβ, especially so in cancer cell physiology. However, specific roles of the LTBPs remain understudied in the context of the primary tumor microenvironment. Herein, we investigated the role of LTBP3 in the distinct processes involved in cancer metastasis. By using three human tumor cell lines of different tissue origin (epidermoid HEp-3 and prostate PC-3 carcinomas and HT-1080 fibrosarcoma) and several metastasis models conducted in both mammalian and avian settings, we show that LTBP3 is involved in the early dissemination of primary cancer cells, namely in the intravasation step of the metastatic cascade. Knockdown of LTBP3 in all tested cell lines led to significant inhibition of tumor cell intravasation, but did not affect primary tumor growth. LTBP3 was dispensable in the late steps of carcinoma cell metastasis that follow tumor cell intravasation, including vascular arrest, extravasation, and tissue colonization. However, LTBP3 depletion diminished the angiogenesis-inducing potential of HEp-3 cells in vivo, which was restorable by exogenous delivery of LTBP3 protein. A similar compensatory approach rescued the dampened intravasation of LTBP3-deficient HEp-3 cells, suggesting that LTBP3 regulates the induction of the intravasation-supporting angiogenic vasculature within developing primary tumors. Using our recently developed microtumor model, we confirmed that LTBP3 loss resulted in the development of intratumoral vessels with an abnormal microarchitecture incompatible with efficient intravasation of HEp-3 carcinoma cells. Collectively, these findings demonstrate that LTBP3 represents a novel oncotarget that has distinctive functions in the regulation of angiogenesis-dependent tumor cell intravasation, a critical process during early cancer dissemination. Our experimental data are also consistent with the survival prognostic value of LTBP3 expression in early-stage head and neck squamous cell carcinomas, further indicating a specific role for LTBP3 in cancer progression toward metastatic disease.

Latent-transforming growth factor beta-binding protein 3 (LTBP-3) is important for craniofacial morphogenesis and hard tissue mineralization, as it is essential for activation of transforming growth factor-beta (TGF-beta). To investigate the role of LTBP-3 in tooth formation we performed micro-computed tomography (micro-CT), histology, and scanning electron microscopy analyses of adult Ltbp3-/- mice. The Ltbp3-/- mutants presented with unique craniofacial malformations and reductions in enamel formation that began at the matrix formation stage. Organization of maturation-stage ameloblasts was severely disrupted. The lateral side of the incisor was affected most. Reduced enamel mineralization, modification of the enamel prism pattern, and enamel nodules were observed throughout the incisors, as revealed by scanning electron microscopy. Molar roots had internal irregular bulbous-like formations. The cementum thickness was reduced, and microscopic dentinal tubules showed minor nanostructural changes. Thus, LTBP-3 is required for ameloblast differentiation and for the formation of decussating enamel prisms, to prevent enamel nodule formation, and for proper root morphogenesis. Also, and consistent with the role of TGF-beta signaling during mineralization, almost all craniofacial bone components were affected in Ltbp3-/- mice, especially those involving the upper jaw and snout. This mouse model demonstrates phenotypic overlap with Verloes Bourguignon syndrome, also caused by mutation of LTBP3, which is hallmarked by craniofacial anomalies and amelogenesis imperfecta phenotypes.

The four latent transforming growth factor-beta (TGF-beta) binding proteins LTBP1-4 are extracellular matrix-associated proteins playing a critical role in the activation of TGF-beta. The LTBP1 gene forms two major transcript variants (i.e. Ltbp1S and Ltbp1L) that are derived from different promoters. We have previously shown the importance of LTBP1 in vivo by using three different Ltbp1 null mice that were either deleted for exons 1 and 2 (Ltbp1L knockout), exon 5 (Ltbp1DeltaEx5), or exon 8 (Ltbp1DeltaEx8). While the Ltbp1L knockout and the Ltbp1DeltaEx8 are perinatal lethal and die of cardiovascular abnormalities, the Ltbp1DeltaEx5 is viable because it expresses a short form of Ltbp1L that lacks 55 amino acids (Delta55 variant of Ltbp1) formed by splicing out exon 5, while lacking the Ltbp1S variant. Since only the Ltbp1DeltaEx5 mouse is viable, we have used this model to address aspects of puberty, fertility, age-dependent reproduction, and ovary function. We report for the first time a function of LTBP1 in female reproduction. The Ltbp1DeltaEx5 females showed impaired fertility associated with delayed sexual maturity (p = 0.0074) and ovarian cyst formation in females older than 40 weeks (p = 0.0204).

Pulsed electromagnetic fields (PEMFs) have been documented to promote bone fracture healing in nonunions and increase lumbar spinal fusion rates. However, the molecular mechanisms by which PEMF stimulates differentiation of human bone marrow stromal cells (hBMSCs) into osteoblasts are not well understood. In this study the PEMF effects on hBMSCs were studied by microarray analysis. PEMF stimulation of hBMSCs' cell numbers mainly affected genes of cell cycle regulation, cell structure, and growth receptors or kinase pathways. In the differentiation and mineralization stages, PEMF regulated preosteoblast gene expression and notably, the transforming growth factor-beta (TGF-beta) signaling pathway and microRNA 21 (miR21) were most highly regulated. PEMF stimulated activation of Smad2 and miR21-5p expression in differentiated osteoblasts, and TGF-beta signaling was essential for PEMF stimulation of alkaline phosphatase mRNA expression. Smad7, an antagonist of the TGF-beta signaling pathway, was found to be miR21-5p's putative target gene and PEMF caused a decrease in Smad7 expression. Expression of Runx2 was increased by PEMF treatment and the miR21-5p inhibitor prevented the PEMF stimulation of Runx2 expression in differentiating cells. Thus, PEMF could mediate its effects on bone metabolism by activation of the TGF-beta signaling pathway and stimulation of expression of miR21-5p in hBMSCs.

Marfan syndrome (MFS) is a multi-system connective tissue disorder that results from mutations to the gene that codes the elastin-associated glycoprotein fibrillin-1. Although elastic fibers are compromised throughout the arterial tree, the most severe phenotype manifests in the ascending aorta. By comparing biaxial mechanics of the ascending and descending thoracic aorta in a mouse model of MFS, we show that aneurysmal propensity correlates well with both a marked increase in circumferential material stiffness and an increase in intramural shear stress despite a near maintenance of circumferential stress. This finding is corroborated via a comparison of the present results with previously reported findings for both the carotid artery from the same mouse model of MFS and for the thoracic aorta from another model of elastin-associated glycoprotein deficiency that does not predispose to thoracic aortic aneurysms. We submit that the unique biaxial loading of the ascending thoracic aorta conspires with fibrillin-1 deficiency to render this aortic segment vulnerable to aneurysm and rupture.

The bioavailability of members of the transforming growth factor beta (TGF-beta) family is controlled by a number of mechanisms. Bona fide TGF-beta is sequestered into the matrix in a latent state and must be activated before it can bind to its receptors. Here, we review the molecules and mechanisms that regulate the bioavailability of TGF-beta and compare these mechanisms with those used to regulate other TGF-beta family members. We also assess the physiological significance of various latent TGF-beta activators, as well as other extracellular modulators of TGF-beta family signaling, by examining the available in vivo data from knockout mouse models and other biological systems.

Prolactinomas are the most frequently observed pituitary adenomas and most of them respond well to conventional treatment with dopamine agonists. However, a subset of prolactinomas fails to respond to such therapies and is considered as dopamine agonist-resistant prolactinomas (DARPs). New therapeutic approaches are necessary for these tumors. TGFbeta1 is a known inhibitor of lactotroph cell proliferation and prolactin secretion, and it partly mediates dopamine inhibitory action. TGFbeta1 is secreted to the extracellular matrix as an inactive latent complex, and its bioavailability is tightly regulated by different components of the TGFbeta1 system including latent binding proteins (LTBPs), local activators (Thrombospondin-1, matrix metalloproteases, integrins, among others), and TGFbeta receptors. Pituitary TGFbeta1 activity and the expression of different components of the TGFbeta1 system, are regulated by dopamine and estradiol. Prolactinomas (animal models and humans) present reduced TGFbeta1 activity as well as reduced expression of several components of the TGFbeta1 system. Therefore, restoration of TGFbeta1 inhibitory activity represents a novel therapeutic approach to bypass dopamine action in DARPs. The aim of this review is to summarize the large literature supporting TGFbeta1 important role as a local modulator of pituitary lactotroph function; as well to provide recent evidence of the restoration of TGFbeta1 activity as an effective treatment in experimental prolactinomas.

Marfan syndrome (MFS) is an autosomal dominant disorder of connective tissue, caused by mutations of the microfibrillar protein fibrillin-1, that predisposes affected individuals to aortic aneurysm and rupture and is associated with increased TGFbeta signaling. TGFbeta is secreted from cells as a latent complex consisting of TGFbeta, the TGFbeta propeptide, and a molecule of latent TGFbeta binding protein (LTBP). Improper extracellular localization of the latent complex can alter active TGFbeta levels, and has been hypothesized as an explanation for enhanced TGFbeta signaling observed in MFS. We previously reported the absence of LTBP-3 in matrices lacking fibrillin-1, suggesting that perturbed TGFbeta signaling in MFS might be due to defective interaction of latent TGFbeta complexes containing LTBP-3 with mutant fibrillin-1 microfibrils. To test this hypothesis, we genetically suppressed Ltbp3 expression in a mouse model of progressively severe MFS. Here, we present evidence that MFS mice lacking LTBP-3 have improved survival, essentially no aneurysms, reduced disruption and fragmentation of medial elastic fibers, and decreased Smad2/3 and Erk1/2 activation in their aortas. These data suggest that, in MFS, improper localization of latent TGFbeta complexes composed of LTBP-3 and TGFbeta contributes to aortic disease progression.

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.