Key Words: heart Ⅲ cardiac fibroblast Ⅲ extracellular matrix Ⅲ Periostin Ⅲ transforming growth factor-␤ signaling A lthough noncardiomyocytes constitute the majority of the cell types present in the postnatal heart and form the cardiac skeleton within which the cardiac myocytes reside, relatively little is known about how the cardiac interstitial microenvironment is formed and the source of the cardiac fibroblast (CF) lineage. The signals that trigger a secretory fibroblast phenotype and collagen formation (fibrogenesis) as well as the morphogenesis of the CF lineage are also not well understood. The CF is the most abundant noncardiomyocyte cell type present within the postnatal mature heart and is chiefly responsible for deposition of the extracellular matrix (ECM). The ECM is considered a dynamic modulatory network because of the continuous changes in secretory activity which alters both cell environment and response throughout development. Indeed, far from inert, the ECM is characterized by constant reorganization in response to endogenous and exogenous stimuli. 1 The ECM also provides structural support for cardiac myocytes and formation of the elaborate cardiac skeleton. The cardiac skeleton encodes the 3D structure of the heart and is composed of a tough sheet of fibrous connective tissue that electrically isolates the atria from the ventricles, contains all four valves and valvular anchorage tissues, and serves as an attachment site for cardiac muscle fibers.Balanced synthesis 2 and degradation 3-5 of this ECM is key to normal cardiovascular development, physiological growth Original

Background-Transforming growth factor-␤ 2 (TGF-␤ 2 ) is a member of a family of growth factors with the potential to modify multiple processes. Mice deficient in the TGF-␤ 2 gene die around birth and show a variety of defects of different organs, including the heart. Methods and Results-We studied the hearts of TGF-␤ 2 -null mouse embryos from 11.5 to 18.5 days of gestation to analyze the types of defects and determine which processes of cardiac morphogenesis are affected by the absence of TGF-␤ 2 . Analysis of serial sections revealed malformations of the outflow tract (typically a double-outlet right ventricle) in 87.5%. There was 1 case of common arterial trunk. Abnormal thickening of the semilunar valves was seen in 4.2%. Associated malformations of the atrioventricular (AV) canal were found in 62.5% and were composed of perimembranous inlet ventricular septal defects (37.5%), AV valve thickening (33.3%), overriding tricuspid valve (25.0%), and complete AV septal defects (4.2%). Anomalies of the aorta and its branches were seen in 33.3%. Immunohistochemical staining showed failure of myocardialization of the mesenchyme of the atrial septum and the ventricular outflow tract as well as deficient valve differentiation. Morphometry documented this to be associated with absence of the normal decrease of total endocardial cushion volume in the older stages. Apoptosis in TGF-␤ 2 -knockout mice was increased, although regional distribution was normal. Conclusions-TGF-␤

Transforming growth factor betas (TGFbetas) are pleiotropic cytokines involved in many biological processes. Genetic engineering and tissue explanation studies have revealed specific non-overlapping roles for TGFbeta ligands and their signaling molecules in development and in normal function of the cardiovascular system in the adult. In the embryo, TGFbetas appear to be involved in epithelial-mesenchymal transformations (EMT) during endocardial cushion formation, and in epicardial epithelial-mesenchymal transformations essential for coronary vasculature, ventricular myocardial development and compaction. In the adult, TGFbetas are involved in cardiac hypertrophy, vascular remodeling and regulation of the renal renin-angiotensin system. The evidence for TGFbeta activities during cardiovascular development and physiologic function will be given and areas which need further investigation will be discussed.

In a common theme of organogenesis, certain cells within a multipotent epithelial sheet exchange signals with their neighbors and develop into a bud structure. Using hair bud morphogenesis as a paradigm, we employed mutant mouse models and cultured keratinocytes to dissect the contributions of multiple extracellular cues in orchestrating adhesion dynamics and proliferation to shape the cluster of cells involved. We found that transforming growth factor β2 signaling is necessary to transiently induce the transcription factor Snail and activate the Ras-mitogen-activated protein kinase (MAPK) pathway in the bud. In the epidermis, Snail misexpression leads to hyperproliferation and a reduction in intercellular adhesion. When E-cadherin is transcriptionally down-regulated, associated adhesion proteins with dual functions in signaling are released from cell-cell contacts, a process which we demonstrate leads to Ras-MAPK activation. These studies provide insights into how multipotent cells within a sheet are stimulated to undergo transcriptional changes that result in proliferation, junctional remodeling, and bud formation. This novel signaling pathway further weaves together the web of different morphogens and downstream transcriptional events that guide hair bud formation within the developing skin.

To examine the roles of TGFbeta isoforms on corneal morphogenesis, the eyes of mice that lack TGFbetas were analyzed at different developmental stages for cell proliferation, migration and apoptosis, and for expression patterns of keratin 12, lumican, keratocan and collagen I. Among the three Tgfb(-/-) mice, only Tgfb2(-/-) mice have abnormal ocular morphogenesis characterized by thin corneal stroma, absence of corneal endothelium, fusion of cornea to lens (a Peters'-like anomaly phenotype), and accumulation of hyaline cells in vitreous. In Tgfb2(-/-) mice, fewer keratocytes were found in stroma that has a decreased accumulation of ECM; for example, lumican, keratocan and collagen I were greatly diminished. The absence of TGFbeta2 did not compromise cell proliferation, nor enhance apoptosis. The thinner stroma resulting from decreased ECM synthesis may account for the decreased cell number in the stroma of Tgfb2 null mice. Keratin 12 expression was not altered in Tgfb2(-/-) mice, implicating normal corneal type epithelial differentiation. Delayed appearance of macrophages in ocular tissues was observed in Tgfb2(-/-) mice. Malfunctioning macrophages may account for accumulation of cell mass in vitreous of Tgfb2 null mice.

The ligand specificity of transforming growth factor beta (TGF␤) in vivo in mouse cardiac cushion epithelial-to-mesenchymal transition (EMT) is poorly understood. To elucidate the function of TGF␤ in cushion EMT, we analyzed Tgfb1 ؊/؊ , Tgfb2 ؊/؊ , and Tgfb3 ؊/؊ mice between embryonic day (E) 9.5 and E14.5 using both in vitro and in vivo approaches. Atrioventricular (AV) canal collagen gel assays at E9.5 indicated normal EMT in both Tgfb1 ؊/؊ and Tgfb3 ؊/؊ mice. However, analysis of Tgfb2 ؊/؊ AV explants at E9.5 and E10.5 indicated that EMT, but not cushion cell proliferation, was initially delayed but later remained persistent. This was concordant with the observation that Tgfb2 ؊/؊ embryos, and not Tgfb1 ؊/؊ or Tgfb3

embryos had an open rostral neural tube, but hematopoiesis and cardiovascular development were ostensibly normal. Golgi membranes of Spca1 ؊/؊ embryos were dilated, had fewer stacked leaflets, and were expanded in amount, consistent with increased Golgi biogenesis. The number of Golgi-associated vesicles was also increased, and rough endoplasmic reticulum had fewer ribosomes. Coated pits, junctional complexes, desmosomes, and basement membranes appeared normal in mutant embryos, indicating that processing and trafficking of proteins in the secretory pathway was not massively impaired. However, apoptosis was increased, possibly the result of secretory pathway stress, and a large increase in cytoplasmic lipid was observed in mutant embryos, consistent with impaired handling of lipid by the Golgi. Adult heterozygous mice appeared normal and exhibited no evidence of Hailey-Hailey disease; however, aged heterozygotes had an increased incidence of squamous cell tumors of keratinized epithelial cells of the skin and esophagus. These data show that loss of the Golgi Ca 2؉ pump causes Golgi stress, expansion of the Golgi, increased apoptosis, and embryonic lethality and demonstrates that SPCA1 haploinsufficiency causes a genetic predisposition to cancer. and Mn 2ϩ (10). Loss of PMR1 affects outer chain glycosylation, proteolytic processing, and trafficking of proteins in the secretory pathway (9). In mammals, SPCA1 is expressed in all tissues (1), whereas SPCA2 is expressed in only a limited set of tissues (3). Like PMR1, both SPCA1 and SPCA2 are localized to the Golgi and transport Ca 2ϩ and Mn 2ϩ (3, 11). There is evidence that the cell biological functions of SPCA1 are also similar to those of PMR1 (12).Loss of one copy of the human ATP2C1 gene, encoding SPCA1, causes Hailey-Hailey disease (HHD), an autosomal dominant skin disorder (13,14). SPCA1 protein levels in HHD keratinocytes are reduced to about half of normal levels, and Golgi Ca 2ϩ handling is impaired (15). HHD is similar to Darier disease, which is caused by null mutations in one copy of the human ATP2A2 gene, encoding SERCA2 (16). Both diseases are characterized by acantholysis (a disruption of cell-cell contacts) in the suprabasal layers of the skin. As the major ER Ca 2ϩ pump in most tissues, including keratinocytes, the function of SERCA2 is similar to that of SPCA1 in that it maintains luminal Ca 2ϩ concentrations in a major compartment of the secretory pathway. In mice, SERCA2 haploinsufficiency does not cause Darier disease but does lead to squamous cell tumors of keratinized epithelial cells (17, 18), the same cell type affected in Darier disease. In humans, a low incidence of squamous cell tumors has been reported in both Darier disease (19) and HHD (20, 21), but it is unclear whether this is a chance association or is caused by the reduction in Ca 2ϩ pump levels and activity. In the current study, we developed a gene-targeted mouse model for SPCA1 and analyzed the phenotype resulting from heterozygous and homozygous null mutations. The results sho...

Exercise improves health and well-being across diverse organ systems, and elucidating mechanisms underlying the beneficial effects of exercise can lead to new therapies. Here, we show that transforming growth factor-β2 (TGF-β2) is secreted from adipose tissue in response to exercise and improves glucose tolerance in mice. We identify TGF-β2 as an exercise-induced adipokine in a gene expression analysis of human subcutaneous adipose tissue biopsies after exercise training. In mice, exercise training increases TGF-β2 in scWAT, serum, and its secretion from fat explants. Transplanting scWAT from exercise-trained wild type mice, but not from adipose tissue-specific Tgfb2−/− mice, into sedentary mice improves glucose tolerance. TGF-β2 treatment reverses the detrimental metabolic effects of high fat feeding in mice. Lactate, a metabolite released from muscle during exercise, stimulates TGF-β2 expression in human adipocytes. Administration of the lactate-lowering agent dichloroacetate during exercise training in mice decreases circulating TGF-β2 levels and reduces exercise-stimulated improvements in glucose tolerance. Thus, exercise training improves systemic metabolism through inter-organ communication with fat via a lactate-TGF-β2-signaling cycle.