GPIHBP1-deficient mice (Gpihbp1؊/؊ ) exhibit severe chylomicronemia. GPIHBP1 is located within capillaries of muscle and adipose tissue, and expression of GPIHBP1 in Chinese hamster ovary cells confers upon those cells the ability to bind lipoprotein lipase (LPL). However, there has been absolutely no evidence that GPIHBP1 actually interacts with LPL in vivo. Heparin is known to release LPL from its in vivo binding sites, allowing it to enter the plasma. After an injection of heparin, we reasoned that LPL bound to GPIHBP1 in capillaries would be released very quickly, and we hypothesized that the kinetics of LPL entry into the plasma would differ in Gpihbp1 ؊/؊ and control mice. Indeed, plasma LPL levels peaked very rapidly (within 1 min) after heparin in control mice. In contrast, plasma LPL levels in Gpihbp1 ؊/؊ mice were much lower 1 min after heparin and increased slowly over 15 min. In keeping with that result, plasma triglycerides fell sharply within 10 min after heparin in wild-type mice, but were negligibly altered in the first 15 min after heparin in Gpihbp1 ؊/؊ mice. Also, an injection of Intralipid released LPL into the plasma of wild-type mice but was ineffective in releasing LPL in Gpihbp1 ؊/؊ mice. The observed differences in LPL release cannot be ascribed to different tissue stores of LPL, as LPL mass levels in tissues were similar in Gpihbp1 ؊/؊ and control mice. The differences in LPL release after intravenous heparin and Intralipid strongly suggest that GPIHBP1 represents an important binding site for LPL in vivo.Triglyceride-rich lipoproteins (chylomicrons and very low density lipoproteins) undergo lipolysis along the surface of capillaries, mainly in heart, skeletal muscle, and adipose tissue (1, 2). This lipolytic processing is carried out by lipoprotein lipase (LPL) 4 (2). LPL is synthesized and secreted by parenchymal cells (e.g. myocytes, adipocytes) and then translocated into the lumen of capillaries, where it is widely assumed to bind to cellsurface heparan sulfate proteoglycans (HSPGs) (1-3). The binding of LPL is thought to involve electrostatic interactions between positively charged "heparin-binding domains" in LPL and negatively charged HSPGs. Multiple lines of evidence have supported this concept. Affinity chromatography of endothelial cell extracts on an LPL-Sepharose column resulted in the isolation of a single 220-kDa heparan sulfate proteoglycan (4). The binding of LPL to cultured endothelial cells can be reduced by removing HSPGs from the surface of cells (5). LPL can be released from its in vivo binding sites by heparin (6), and mutation of the principal heparin-binding domain of LPL reduces LPL binding to cells (7).Recent findings have suggested that the paradigm for lipolysis of lipoproteins by LPL requires updating (8 -11). Adult mice lacking glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (Gpihbp1 Ϫ/Ϫ ) exhibit chylomicronemia (8), a severe form of hypertriglyceridemia that, in humans, is often caused by a deficiency in LPL (2). GPIHBP1 i...

Smad proteins are intracellular molecules that mediate the canonical signaling cascade of TGFβ superfamily growth factors. The TGFβ superfamily comprises two groups of growth factors, BMPs and TGFβs. Both groups can be further divided into several sub-groups based on sequence homologies and functional similarities. Ligands of the TGFβ superfamily bind to cell surface receptors to activate Smad proteins in the cytoplasm; then the activated Smad proteins translocate into the nucleus to activate or repress specific target gene transcription. Both groups of growth factors play important roles in skeletal development and regeneration. However, whether these effects reflect signaling through canonical Smad pathways, or other non-canonical signaling pathways in vivo remains a mystery. Moreover, the mechanisms utilized by Smad proteins to initiate nuclear events and their interactions with cytoplasmic proteins are still under intensive investigation. This review will discuss the most recent progress understanding Smad signaling in the context of skeletal development and regeneration.

Bone morphogenetic protein (BMP) signaling pathways regulate multiple aspects of endochondral bone formation. The importance of extracellular antagonists as regulators of BMP signaling has been defined. In vitro studies reveal that the intracellular regulators, inhibitory Smads 6 and 7, can regulate BMP-mediated effects on chondrocytes. Although in vivo studies in which inhibitory Smads were overexpressed in cartilage have shown that inhibitory Smads have the potential to limit BMP signaling in vivo, the physiological relevance of inhibitory Smad activity in skeletal tissues is unknown. In this study, we have determined the role of Smad6 in endochondral bone formation. Loss of Smad6 in mice leads to defects in both axial and appendicular skeletal development. Specifically, Smad6−/− mice exhibit a posterior transformation of the seventh cervical vertebra, bilateral ossification centers in lumbar vertebrae, and bifid sternebrae due to incomplete sternal band fusion. Histological analysis of appendicular bones revealed delayed onset of hypertrophic differentiation and mineralization at midgestation in Smad6−/− mice. By late gestation, however, an expanded hypertrophic zone, associated with an increased pool of proliferating cells undergoing hypertrophy, was evident in Smad6 mutant growth plates. The mutant phenotype is attributed, at least in part, to increased BMP responsiveness in Smad6-deficient chondrocytes. Overall, our results show that Smad6 is required to limit BMP signaling during endochondral bone formation.

Fibroblast growth factor receptor 2 (FGFR2) is a crucial regulator of bone formation during embryonic development. Both gain and loss-of-function studies in mice have shown that FGFR2 maintains a critical balance between the proliferation and differentiation of osteoprogenitor cells. We have identified de novo FGFR2 mutations in a sporadically occurring perinatal lethal skeletal dysplasia characterized by poor mineralization of the calvarium, craniosynostosis, dysmorphic facial features, prenatal teeth, hypoplastic pubis and clavicles, osteopenia, and bent long bones. Histological analysis of the long bones revealed that the growth plate contained smaller hypertrophic chondrocytes and a thickened hypercellular periosteum. Four unrelated affected individuals were found to be heterozygous for missense mutations that introduce a polar amino acid into the hydrophobic transmembrane domain of FGFR2. Using diseased chondrocytes and a cell-based assay, we determined that these mutations selectively reduced plasma-membrane levels of FGFR2 and markedly diminished the receptor's responsiveness to extracellular FGF. All together, these clinical and molecular findings are separate from previously characterized FGFR2 disorders and represent a distinct skeletal dysplasia.

Our results demonstrate that ventricular wall cardiomyocyte proliferation is subdivided into distinct regulatory phases. Each involves instructive cues that originate outside the heart and thereby act on the epicardium in an endocrine manner, a mode of regulation that is mostly unknown in embryogenesis.