The extracellular matrix protein MAGP1 supports thermogenesis and protects against obesity and diabetes through regulation of TGF-ß.

Microfibril-associated glycoprotein 1 (MAGP1) is a component of extracellular matrix microfibrils. Here we show that MAGP1 expression is significantly altered in obese humans, and inactivation of the MAGP1 gene (Mfap2(-/-)) in mice results in adipocyte hypertrophy and predisposition to metabolic dysfunction. Impaired thermoregulation was evident in Mfap2(-/-) mice prior to changes in adiposity, suggesting a causative role for MAGP1 in the increased adiposity and predisposition to diabetes. By 5 weeks of age, Mfap2(-/-) mice were maladaptive to cold challenge, uncoupling protein-1 expression was attenuated in the brown adipose tissue, and there was reduced browning of the subcutaneous white adipose tissue. Levels of transforming growth factor-ß (TGF-ß) activity were elevated in Mfap2(-/-) adipose tissue, and the treatment of Mfap2(-/-) mice with a TGF-ß-neutralizing antibody improved their body temperature and prevented the increased adiposity phenotype. Together, these findings indicate that the regulation of TGF-ß by MAGP1 is protective against the effects of metabolic stress, and its absence predisposes individuals to metabolic dysfunction.

Microfibril-associated glycoprotein 2 (MAGP2) loss of function has pleiotropic effects in vivo.

Microfibril-associated glycoprotein (MAGP) 1 and 2 are evolutionarily related but structurally divergent proteins that are components of microfibrils of the extracellular matrix. Using mice with a targeted inactivation of Mfap5, the gene for MAGP2 protein, we demonstrate that MAGPs have shared as well as unique functions in vivo. Mfap5(-/-) mice appear grossly normal, are fertile, and have no reduction in life span. Cardiopulmonary development is typical. The animals are normotensive and have vascular compliance comparable with age-matched wild-type mice, which is indicative of normal, functional elastic fibers. Loss of MAGP2 alone does not significantly alter bone mass or architecture, and loss of MAGP2 in tandem with loss of MAGP1 does not exacerbate MAGP1-dependent osteopenia. MAGP2-deficient mice are neutropenic, which contrasts with monocytopenia described in MAGP1-deficient animals. This suggests that MAGP1 and MAGP2 have discrete functions in hematopoiesis. In the cardiovascular system, MAGP1;MAGP2 double knockout mice (Mfap2(-/-);Mfap5(-/-)) show age-dependent aortic dilation. These findings indicate that MAGPs have shared primary functions in maintaining large vessel integrity. In solid phase binding assays, MAGP2 binds active TGFß1, TGFß2, and BMP2. Together, these data demonstrate that loss of MAGP2 expression in vivo has pleiotropic effects potentially related to the ability of MAGP2 to regulate growth factors or participate in cell signaling.

Pod1/Tcf21 is regulated by retinoic acid signaling and inhibits differentiation of epicardium-derived cells into smooth muscle in the developing heart.

Epicardium-derived cells (EPDCs) invade the myocardium and differentiate into fibroblasts and vascular smooth muscle (SM) cells, which support the coronary vessels. The transcription factor Pod1 (Tcf21) is expressed in subpopulations of the epicardium and EPDCs in chicken and mouse embryonic hearts, and the transcription factors WT1, NFATC1, and Tbx18 are expressed in overlapping and distinct subsets of Pod1-expressing cells. Expression of Pod1 and WT1, but not Tbx18 or NFATC1, is activated with all-trans-retinoic acid (RA) treatment of isolated chick EPDCs in culture. In intact chicken hearts, RA inhibition leads to decreased Pod1 expression while RA treatment inhibits SM differentiation. The requirements for Pod1 in differentiation of EPDCs in the developing heart were examined in mice lacking Pod1. Loss of Pod1 in mice leads to epicardial blistering, increased SM differentiation on the surface of the heart, and a paucity of interstitial fibroblasts, with neonatal lethality. Epicardial epithelial-to-mesenchymal transition (EMT) and endothelial differentiation of coronary vessels are relatively unaffected. On the surface of the myocardium, expression of multiple SM markers is increased in Pod1-deficient EPDCs, demonstrating premature SM differentiation. Increased SM differentiation also is observed in Pod1-deficient lung mesenchyme. Together, these data demonstrate a critical role for Pod1 in controlling mesenchymal progenitor cell differentiation into SM and fibroblast lineages during cardiac development.

NFATC1 promotes epicardium-derived cell invasion into myocardium.

Epicardium-derived cells (EPDCs) contribute to formation of coronary vessels and fibrous matrix of the mature heart. Nuclear factor of activated T-cells cytoplasmic 1 (NFATC1) is expressed in cells of the proepicardium (PE), epicardium and EPDCs in mouse and chick embryos. Conditional loss of NFATC1 expression in EPDCs in mice causes embryonic death by E18.5 with reduced coronary vessel and fibrous matrix penetration into myocardium. In osteoclasts, calcineurin-mediated activation of NFATC1 by receptor activator of NFκB ligand (RANKL) signaling induces cathepsin K (CTSK) expression for extracellular matrix degradation and cell invasion. RANKL/NFATC1 pathway components also are expressed in EPDCs, and loss of NFATC1 in EPDCs causes loss of CTSK expression in the myocardial interstitium in vivo. Likewise, RANKL treatment induces Ctsk expression in PE-derived cell cultures via a calcineurin-dependent mechanism. In chicken embryo hearts, RANKL treatment increases the distance of EPDC invasion into myocardium, and this response is calcineurin dependent. Together, these data demonstrate a crucial role for the RANKL/NFATC1 signaling pathway in promoting invasion of EPDCs into the myocardium by induction of extracellular matrix-degrading enzyme gene expression.

Transcriptional regulation of heart valve progenitor cells.

The development and normal function of the heart valves requires complex interactions among signaling molecules, transcription factors and structural proteins that are tightly regulated in time and space. Here we review the roles of critical transcription factors that are required for specific aspects of normal valve development. The early progenitors of the heart valves are localized in endocardial cushions that express transcription factors characteristic of mesenchyme, including Twist1, Tbx20, Msx1 and Msx2. As the valve leaflets mature, they are composed of complex stratified extracellular matrix proteins that are regulated by the transcriptional functions of NFATc1, Sox9, and Scleraxis. Each of these factors has analogous functions in differentiation of related connective tissue lineages. Together, the precise timing and localized functions of specific transcription factors control cell proliferation, differentiation, elongation, and remodeling processes that are necessary for normal valve structure and function. In addition, there is increasing evidence that these same transcription factors contribute to congenital, as well as degenerative, valve disease.

VEGF and RANKL regulation of NFATc1 in heart valve development.

RATIONALE: NFATc1 (nuclear factor of activated T-cells cytoplasmic 1) activity in endocardial cushion (ECC) endothelial cells is required for normal ECC growth and extracellular matrix (ECM) remodeling during heart valve development. OBJECTIVE: The mechanisms of NFATc1 activation and downstream effects on cell proliferation and ECM-remodeling enzyme gene expression were examined in NFATc1 mutant mice and chick ECC explants. METHODS AND RESULTS: NFATc1(-/-) mice display reduced proliferation of ECC endothelial and mesenchymal cells at embryonic day 10.5, whereas myocardial cells are unaffected. Vascular endothelial growth factor A (VEGF) activates NFATc1 and promotes ECC cell proliferation via the regulatory phosphatase, calcineurin, and mitogen-activated protein kinase-extracellular signal-regulated kinase 1-extracellular signal-regulated kinase 1/2 (MEK1-ERK1/2)-dependent signaling. As ECCs mature, RANKL (receptor activator of nuclear factor kappaB ligand) and the ECM-remodeling enzyme cathepsin K (CtsK) are expressed by ECC endothelial cells. RANKL inhibits VEGF-induced cell proliferation while causing increased expression of CtsK via calcineurin/NFATc1 and c-Jun N-terminal kinase (JNK)1/2-dependent signaling. CONCLUSION: These data support a novel mechanism for the transition from ECC growth to remodeling in which NFATc1 promotes a sequential pattern of gene expression via cooperation with ligand-specific cofactors such as MEK1-ERK1/2 or JNK1/2.

Heart valve development: regulatory networks in development and disease.

In recent years, significant advances have been made in the definition of regulatory pathways that control normal and abnormal cardiac valve development. Here, we review the cellular and molecular mechanisms underlying the early development of valve progenitors and establishment of normal valve structure and function. Regulatory hierarchies consisting of a variety of signaling pathways, transcription factors, and downstream structural genes are conserved during vertebrate valvulogenesis. Complex intersecting regulatory pathways are required for endocardial cushion formation, valve progenitor cell proliferation, valve cell lineage development, and establishment of extracellular matrix compartments in the stratified valve leaflets. There is increasing evidence that the regulatory mechanisms governing normal valve development also contribute to human valve pathology. In addition, congenital valve malformations are predominant among diseased valves replaced late in life. The understanding of valve developmental mechanisms has important implications in the diagnosis and management of congenital and adult valve disease.