Two novel compound heterozygous variants of LTBP4 in a Chinese infant with cutis laxa type IC and a review of the related literature.

BACKGROUND: Autosomal recessive cutis laxa type IC (ARCL IC, MIM: #613177) results from a mutation in the LTBP4 gene (MIM: #604710) on chromosome 19q13. CASE PRESENTATION: A 28-day-old Chinese infant with generalized cutis laxa accompanied by impaired pulmonary, gastrointestinal, genitourinary, retinal hemorrhage, abnormality of coagulation and hyperbilirubinemia was admitted to our hospital. To find out the possible causes of these symptoms, whole-exome sequencing was performed on the infant. Two novel pathogenic frame-shift variants [c.605_606delGT (p.Ser204fs * 8) and c.1719delC (p.Arg574fs * 199)] of the LTBP4 gene associated with ARCL IC were found which was later verified by Sanger sequencing. The pathogenicity of mutations was subsequently assessed by several software programs and databases. In addition, an analytical review on the clinical phenotypes of the disease previously reported in literature was performed. CONCLUSIONS: This is the first report of a Chinese infant with ARCL IC in China due to novel pathogenic variations of LTBP4. Our study extends the cutis laxa type IC mutation spectrum as well as the phenotypes associated with the disease in different populations.

LTBP3 promotes early metastatic events during cancer cell dissemination.

Latent transforming growth factor ß (TGFß)-binding proteins (LTBPs) are important for the secretion, activation, and function of mature TGFß, 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.

Genetic modifiers of muscular dystrophy act on sarcolemmal resealing and recovery from injury.

Genetic disruption of the dystrophin complex produces muscular dystrophy characterized by a fragile muscle plasma membrane leading to excessive muscle degeneration. Two genetic modifiers of Duchenne Muscular Dystrophy implicate the transforming growth factor ß (TGFß) pathway, osteopontin encoded by the SPP1 gene and latent TGFß binding protein 4 (LTBP4). We now evaluated the functional effect of these modifiers in the context of muscle injury and repair to elucidate their mechanisms of action. We found that excess osteopontin exacerbated sarcolemmal injury, and correspondingly, that loss of osteopontin reduced injury extent both in isolated myofibers and in muscle in vivo. We found that ablation of osteopontin was associated with reduced expression of TGFß and TGFß-associated pathways. We identified that increased TGFß resulted in reduced expression of Anxa1 and Anxa6, genes encoding key components of the muscle sarcolemma resealing process. Genetic manipulation of Ltbp4 in dystrophic muscle also directly modulated sarcolemmal resealing, and Ltbp4 alleles acted in concert with Anxa6, a distinct modifier of muscular dystrophy. These data provide a model in which a feed forward loop of TGFß and osteopontin directly impacts the capacity of muscle to recover from injury, and identifies an intersection of genetic modifiers on muscular dystrophy.

Role of LTBP4 in alveolarization, angiogenesis, and fibrosis in lungs.

Deficiency of the extracellular matrix protein latent transforming growth factor-ß (TGF-ß)-binding protein-4 (LTBP4) results in lack of intact elastic fibers, which leads to disturbed pulmonary development and lack of normal alveolarization in humans and mice. Formation of alveoli and alveolar septation in pulmonary development requires the concerted interaction of extracellular matrix proteins, growth factors such as TGF-ß, fibroblasts, and myofibroblasts to promote elastogenesis as well as vascular formation in the alveolar septae. To investigate the role of LTBP4 in this context, lungs of LTBP4-deficient (Ltbp4-/-) mice were analyzed in close detail. We elucidate the role of LTBP4 in pulmonary alveolarization and show that three different, interacting mechanisms might contribute to alveolar septation defects in Ltbp4-/- lungs: 1) absence of an intact elastic fiber network, 2) reduced angiogenesis, and 3) upregulation of TGF-ß activity resulting in profibrotic processes in the lung.

Hepatic cyclooxygenase-2 overexpression induced spontaneous hepatocellular carcinoma formation in mice.

Cyclooxygenase (COX)-2 is upregulated in hepatocellular carcinoma (HCC). However, the direct causative effect of COX-2 in spontaneous HCC formation remains unknown. We thus investigate the role and molecular pathogenesis of COX-2 in HCC by using liver-specific COX-2 transgenic (TG) mice. We found spontaneous HCC formation with elevated inflammatory infiltrates and neovessels in male TG mice (3/21, 14.3%), but not in any of male WT mice (0/19). Reduced representation bisulfite sequencing (RRBS) and gene expression microarrays were performed in the HCC tumor and non-HCC liver tissues to investigate the molecular mechanisms of COX-2-driven HCC. By RRBS, DNA promoter hypermethylation was identified in HCC from TG mice. Induction of promoter hypermethylation was associated with reduced tet methylcytosine dioxygenase 1 (TET1) expression by COX-2. TET1 could catalyze the conversion of 5-methylcytosine into 5-hydroxymethylcytosine (5hmC) and prevents DNA hypermethylation. In keeping with this, loss of 5hmC was demonstrated in COX-2-induced HCC. Consistently, COX-2 overexpression in human HCC cell lines could reduce both TET1 expression and 5hmc levels. Integrative analyses of DNA methylation and gene expression profiles further identified significantly downregulated genes including LTBP1, ADCY5 and PRKCZ by promoter methylation in COX-2-induced HCC. Reduced expression of LTBP1, ADCY5 and PRKCZ by promoter hypermethylation was further validated in human HCCs. Bio-functional investigation revealed that LTBP1 inhibited cell proliferation in HCC cell lines, suggesting its potential role as a tumor suppressor in HCC. Gene expression microarrays revealed that signaling cascades (AKT (protein kinase B), STK33 (Serine/Threonine kinase 33) and MTOR (mechanistic target of rapamycin) pathways) were enriched in COX-2-induced HCC. In conclusion, this study demonstrated for the first time that enhanced COX-2 expression in hepatocytes is sufficient to induce HCC through inducing promoter hypermethylation by reducing TET1, silencing tumor-suppressive genes and activating key oncogenic pathways. Inhibition of COX-2 represents a mechanism-based target for HCC prevention.

A Mutation in LTBP2 Causes Congenital Glaucoma in Domestic Cats (Felis catus).

The glaucomas are a group of diseases characterized by optic nerve damage that together represent a leading cause of blindness in the human population and in domestic animals. Here we report a mutation in LTBP2 that causes primary congenital glaucoma (PCG) in domestic cats. We identified a spontaneous form of PCG in cats and established a breeding colony segregating for PCG consistent with fully penetrant, autosomal recessive inheritance of the trait. Elevated intraocular pressure, globe enlargement and elongated ciliary processes were consistently observed in all affected cats by 8 weeks of age. Varying degrees of optic nerve damage resulted by 6 months of age. Although subtle lens zonular instability was a common feature in this cohort, pronounced ectopia lentis was identified in less than 10% of cats examined. Thus, glaucoma in this pedigree is attributed to histologically confirmed arrest in the early post-natal development of the aqueous humor outflow pathways in the anterior segment of the eyes of affected animals. Using a candidate gene approach, significant linkage was established on cat chromosome B3 (LOD 18.38, θ = 0.00) using tightly linked short tandem repeat (STR) loci to the candidate gene, LTBP2. A 4 base-pair insertion was identified in exon 8 of LTBP2 in affected individuals that generates a frame shift that completely alters the downstream open reading frame and eliminates functional domains. Thus, we describe the first spontaneous and highly penetrant non-rodent model of PCG identifying a valuable animal model for primary glaucoma that closely resembles the human disease, providing valuable insights into mechanisms underlying the disease and a valuable animal model for testing therapies.

Latent TGF-ß-binding proteins.

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

Function of latent TGFß binding protein 4 and fibulin 5 in elastogenesis and lung development.

Mice deficient in Latent TGFß Binding Protein 4 (Ltbp4) display a defect in lung septation and elastogenesis. The lung septation defect is normalized by genetically decreasing TGFß2 levels. However, the elastic fiber assembly is not improved in Tgfb2(-/-) ;Ltbp4S(-/-) compared to Ltbp4S(-/-) lungs. We found that decreased levels of TGFß1 or TGFß3 did not improve lung septation indicating that the TGFß isoform elevated in Ltbp4S(-/-) lungs is TGFß2. Expression of a form of Ltbp4 that could not bind latent TGFß did not affect lung phenotype indicating that normal lung development does not require the formation of LTBP4-latent TGFß complexes. Therefore, the change in TGFß-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 TGFß2 in lung septation and for Ltbp4 in regulating fibulin-5 dependent elastic fiber assembly.

Cerebral small vessel disease-related protease HtrA1 processes latent TGF-ß binding protein 1 and facilitates TGF-ß signaling.

High temperature requirement protein A1 (HtrA1) is a primarily secreted serine protease involved in a variety of cellular processes including transforming growth factor ß (TGF-ß) signaling. Loss of its activity causes cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL), an inherited form of cerebral small vessel disease leading to early-onset stroke and premature dementia. Dysregulated TGF-ß signaling is considered to promote CARASIL pathogenesis, but the underlying molecular mechanisms are incompletely understood. Here we present evidence from mouse brain tissue and embryonic fibroblasts as well as patient skin fibroblasts for a facilitating role of HtrA1 in TGF-ß pathway activation. We identify latent TGF-ß binding protein 1 (LTBP-1), an extracellular matrix protein and key regulator of TGF-ß bioavailability, as a novel HtrA1 target. Cleavage occurs at physiological protease concentrations, is prevented under HtrA1-deficient conditions as well as by CARASIL mutations and disrupts both LTBP-1 binding to fibronectin and its incorporation into the extracellular matrix. Hence, our data suggest an attenuation of TGF-ß signaling caused by a lack of HtrA1-mediated LTBP-1 processing as mechanism underlying CARASIL pathogenesis.

Zebrafish second heart field development relies on progenitor specification in anterior lateral plate mesoderm and nkx2.5 function.

Second heart field (SHF) progenitors perform essential functions during mammalian cardiogenesis. We recently identified a population of cardiac progenitor cells (CPCs) in zebrafish expressing latent TGFß-binding protein 3 (ltbp3) that exhibits several defining characteristics of the anterior SHF in mammals. However, ltbp3 transcripts are conspicuously absent in anterior lateral plate mesoderm (ALPM), where SHF progenitors are specified in higher vertebrates. Instead, ltbp3 expression initiates at the arterial pole of the developing heart tube. Because the mechanisms of cardiac development are conserved evolutionarily, we hypothesized that zebrafish SHF specification also occurs in the ALPM. To test this hypothesis, we Cre/loxP lineage traced gata4(+) and nkx2.5(+) ALPM populations predicted to contain SHF progenitors, based on evolutionary conservation of ALPM patterning. Traced cells were identified in SHF-derived distal ventricular myocardium and in three lineages in the outflow tract (OFT). We confirmed the extent of contributions made by ALPM nkx2.5(+) cells using Kaede photoconversion. Taken together, these data demonstrate that, as in higher vertebrates, zebrafish SHF progenitors are specified within the ALPM and express nkx2.5. Furthermore, we tested the hypothesis that Nkx2.5 plays a conserved and essential role during zebrafish SHF development. Embryos injected with an nkx2.5 morpholino exhibited SHF phenotypes caused by compromised progenitor cell proliferation. Co-injecting low doses of nkx2.5 and ltbp3 morpholinos revealed a genetic interaction between these factors. Taken together, our data highlight two conserved features of zebrafish SHF development, reveal a novel genetic relationship between nkx2.5 and ltbp3, and underscore the utility of this model organism for deciphering SHF biology.

Latent TGF-ß 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-ß 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-ß because TGF-ß-free recombinant LTBP-4 was as potent an elastogenic inducer as TGF-ß-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.

Long form of latent TGF-ß binding protein 1 (Ltbp1L) regulates cardiac valve development.

Transforming Growth Factor ß (TGF-ß) is crucial for valve development and homeostasis. The long form of Latent TGF-ß binding protein 1 (LTBP1L) covalently binds all TGF-ß isoforms and regulates their bioavailability. Ltbp1L expression analysis during valvulogenesis revealed two patterns of Ltbp1L production: an early one (E9.5-11.5) associated with endothelial-to-mesenchymal transformation (EMT); and a late one (E12.5 to birth) contemporaneous with valve remodeling. Similarly, histological analysis of Ltbp1L(-/-) developing valves identified two different pathologies: generation of hypoplastic endocardial cushions in early valvulogenesis, followed by development of hyperplastic valves in late valvulogenesis. Ltbp1L promotes valve EMT, as Ltbp1L absence yields hypoplastic endocardial cushions in vivo and attenuated EMT in vitro. Ltbp1L(-/-) valve hyperplasia in late valvuogenesis represents a consequence of prolonged EMT. We demonstrate that Ltbp1L is a major regulator of Tgf-ß activity during valvulogenesis since its absence results in a perturbed Tgf-ß pathway that causes all Ltbp1L(-/-) valvular defects.

LTBP-2 has multiple heparin/heparan sulfate binding sites.

Latent transforming growth factor-beta-1 binding protein-2 (LTBP-2) is a protein of poorly understood function associated with fibrillin-1-containing microfibrils during elastinogenesis. In this study we investigated the molecular interactions of LTBP-2 with heparin and heparan sulfate proteoglycans (HSPGs) since unidentified cell surface HSPGs are critical for normal fiber assembly. In solid phase assays, heparin conjugated to albumin (HAC) bound strongly to recombinant full-length human LTBP-2. This interaction was completely blocked by addition of excess heparin, but not chondroitin sulfate, confirming specificity. Analysis of binding to LTBP-2 fragments showed that HAC bound strongly to N-terminal fragment LTBP-2 NT(H) and more weakly to central fragment LTBP-2 C(H). No binding was detected to C-terminal fragment LTBP-2 CT(H). Kds for heparin binding were calculated for full-length LTBP-2, LTBP-2 NT(H) and LTBP-2 C(H) as 0.9 nM, 0.7 nM and 80 nM respectively. HAC interaction with fragment LTBP-2 NT(H) was not sensitive to EDTA or EGTA indicating that binding had no requirement for Ca(2+) ions whereas HAC binding to fragment LTBP-2 C(H) was markedly reduced by these chelating agents indicating a degree of Ca(2+) dependence. Inhibition studies with synthetic peptides identified three major heparin binding sequences in fragment LTBP-2 NT(H), including sequence LTEKIKKIKIV in the first large cysteine-free domain of LTBP-2, adjacent to the previously identified fibulin-5 binding site. LTBP-2 was found to interact strongly in a heparin-inhibitable manner with cell surface HSPG syndecan-4, but showed no interaction with recombinant syndecan-2. LTBP-2 also showed strong interaction with the heparan sulfate chains of basement membrane HSPG, perlecan. The potential importance of HSPG-LTBP-2 interactions in elastic fiber assembly and microfibril attachment to basement membranes is discussed.

Latent TGF-beta binding proteins (LTBPs)-1 and -3 coordinate proliferation and osteogenic differentiation of human mesenchymal stem cells.

Mesenchymal stem cells (MSCs) possess the capability to differentiate into bone forming cells, osteoblasts, and thus represent a new therapeutic tool in regenerative medicine. Transforming growth factor (TGF)-beta is abundantly present in bone tissue where it regulates osteoblast and osteoclast functions in a complex manner. Latent TGF-beta binding protein (LTBP)-1 mediates the extracellular matrix (ECM) targeting and accumulation of most TGF-beta in the bone. We describe here an important regulatory role for LTBP-3 in TGF-beta activation and autocrine growth control in MSCs. LTBP-3 knockdown via siRNA mediated silencing resulted in reduced cell proliferation and reduced osteogenic differentiation. When MSCs were induced to undergo differentiation, LTBP-3 levels became downregulated in parallel with reduced TGF-beta activation. These changes coincided with the matrix maturation phase of osteogenic differentiation. The mechanism of LTBP-3 is most likely via TGF-beta activation in the early proliferative phase of the differentiation process. Later, when TGF-beta activity would inhibit further maturation and mineralization, LTBP-3 expression becomes downregulated and LTBP-1 containing large latent TGF-beta1 complexes accumulate into the ECM. These complexes represent readily available targets for osteoclast mediated release and activation of TGF-beta in bone tissue. Our results provide evidence that LTBP isoforms can differentially regulate TGF-beta activation and ECM accumulation during osteogenic differentiation.

Fibronectin and heparin binding domains of latent TGF-beta binding protein (LTBP)-4 mediate matrix targeting and cell adhesion.

Latent transforming growth factor (TGF)-beta binding proteins are extracellular matrix (ECM) proteins involved in the regulation of TGF-beta sequestration and activation. In this study, we have identified binding domains in LTBP-4, which mediate matrix targeting and cell adhesion. LTBP-4 was found to possess heparin binding activity, especially in its N-terminal region. The C-terminal domain of LTBP-4 supported fibroblast adhesion, a property reduced by soluble heparin. In addition, we found that LTBP-4 binds directly to fibronectin (FN), which was indispensable for the matrix assembly of LTBP-4. The FN binding sites were also located in the N-terminal region. Interestingly, heparin was able to reduce the binding of LTBP-4 to FN. In fibroblast cultures, LTBP-4 colocalized first with FN and subsequently with fibrillin-1, pointing to a role for FN in the early assembly of LTBP-4. In FN -/- fibroblasts, LTBP-mediated ECM targeting was disturbed, resulting in increased TGF-beta activity. These results revealed new molecular interactions which are evidently important for the ECM targeting, but which also are evidence of novel functions for LTBP-4 as an adhesion molecule.

[Functional histology of dermis]. / Histologie fonctionnelle du derme.

The skin is composed of epidermis, dermis and subcutaneous tissue that interconnect anatomically. The dermis is an integrated system of fibrous and amorphous connective tissue that accommodates nerve and vascular networks, epidermally derived appendages, fibroblasts, macrophages and mast cells. Elastic and collagen tissue are the main types of fibrous connective tissue. The elastic connective tissue is assembled in a continuous network including mature elastic fibers, immature elaunin fibers and oxytalan fibers. Mature elastic fibers and elaunin have microfibrillar and amorphous matrix components while oxytalan fibers only contain microfibrils. Several molecules have been identified as constituents of the elastic fibers. Among the most characterized of these molecules is elastin in amorphous matrix, fibrillins 1 and 2 and LTBP-2 (ligand of latent TGFbeta) in microfibrils and fibulins which interconnect elastin and fibrillins. Elastic fibers provides elasticity to the skin. Under electron microscope, collagen fibers appears as of bundles of periodically banded fibrils which are composed of collagens types I, III and V; type V collagen is believed to assist in regulating fibril diameter. They are associated with FACITs (fibril-associated collagen with interrupted triple helixes) collagens types XIV et XVI. Collagen fibers provide tensile strength to the skin. Non fibrous connective tissue molecules include finely filamentous glycoproteins, glycosaminoglycans and proteoglycans of "the ground substance" (hyaluronic acid and chondroitin sulphate, dermatan sulphate, versican, decorin). Fibroblasts, macrophages and mast cells are regular residents of the dermis. The main function of these cells are well known. Fibroblasts are responsible for the synthesis and the degradation of fibrous and non fibrous connective tissue matrix proteins. Macrophages are phagocytic; they process and present antigen to immunocompetent lymphoid cells. Mast cells are responsible for IgE mediated acute, subacute and chronic inflammation. All these cells have a long list of other functions, in particular they are involved in coagulation, wound healing and tissue remodeling.