Cx43 in osteogenic cells controls both arms of the bone-remodeling cycle via direct actions on osteoblast differentiation and function and indirect modulation of osteoclastogenesis. These result in changes remindful of those that occur in skeletal disuse or aging and disclose a far broader function of Cx43 in skeletal biology.

Objective-Lipin 1 controls fatty acid metabolism in the nucleus as a transcriptional regulator and in the cytosol as an enzyme catalyzing the penultimate step in phosphoglycerol triacylglyceride (TAG) synthesis. We sought to evaluate the effects of lipin 1 on hepatic TAG synthesis and secretion by gain-of-function and loss-of-function approaches. Methods and Results-Rates of TAG synthesis were not impaired in hepatocytes isolated from adult lipin 1-deficient (fld) mice and were actually increased in 14-day-old fld mice. Additionally, compared to littermate controls, VLDL-TAG secretion rates were markedly increased in fld mice of both ages. Lipin 1 overexpression did not alter TAG synthesis rates but significantly suppressed VLDL-TAG secretion. The lipin 1-mediated suppression of VLDL-TAG secretion was linked to the peptide motif mediating its transcriptional-regulatory effects. However, the expression of candidate genes required for VLDL assembly and secretion was unaltered by lipin 1 activation or deficiency. Finally, the hepatic expression of lipin 1 was diminished in obese insulin-resistant mice, whereas adenoviral-mediated overexpression of lipin 1 in liver of these mice inhibits VLDL-TAG secretion and improves hepatic insulin signaling. Conclusions-Collectively, these studies reveal new and unexpected effects of lipin 1 on hepatic TAG metabolism and obesity-related hepatic insulin resistance.

The gap junction protein, connexin43 (Cx43) controls both bone formation and osteoclastogenesis via osteoblasts and/or osteocytes. Cx43 has also been proposed to mediate an anti-apoptotic effect of bisphosphonates, potent inhibitors of bone resorption. We studied whether bisphosphonates are effective in protecting mice with a conditional Cx43 gene deletion in osteoblasts and osteocytes (cKO) from the consequences of ovariectomy on bone mass and strength. Ovariectomy resulted in rapid loss of trabecular bone followed by a slight recovery in wild type (WT) mice, and a similar degree of trabecular bone loss, albeit slightly delayed, occurred in cKO mice. Treatment with either risedronate (20µg/kg) or alendronate (40µg/kg) prevented ovariectomy-induced bone loss in both genotypes. In basal conditions, bones of cKO mice have larger marrow area, higher endocortical osteoclast number, and lower cortical thickness and strength relative to WT. Ovariectomy increased endocortical osteoclast number in WT but not in cKO mice. Both bisphosphonates prevented these increases in WT mice, and normalized endocortical osteoclast number, cortical thickness and bone strength in cKO mice. Thus, lack of osteoblast/osteocyte Cx43 does not alter bisphosphonate action on bone mass and strength in estrogen deficiency. These results support the notion that one of the main functions of Cx43 in cortical bone is to restrain osteoblast and/or osteocytes from inducing osteoclastogenesis at the endocortical surface.

Microsomal triglyceride transfer protein (Mttp) is a key player in the assembly and secretion of hepatic very low density lipoproteins (VLDL). Here we determined the effects of Mttp overexpression on hepatic triglyceride (TG) and VLDL secretion in leptin-deficient (ob/ob) mice, specifically in relation to apolipoproteinB (apoB) isoforms. We crossed Apobec1 2/2 mice with congenic ob/ob mice to generate apoB100-only ob/ob mice (A-ob/ob). The obesity phenotype in both genotypes was similar, but A-ob/ob mice had greater hepatic TG content. Administration of recombinant adenovirus expressing murine Mttp cDNA (Ad-mMTP) increased hepatic Mttp content and activity and increased hepatic VLDL-TG secretion in A-ob/ob mice. However, despite equivalent overexpression of Mttp, there was no change in VLDL-TG secretion in ob/ob mice in a wild-type Apobec1 background. Metabolic labeling studies in primary hepatocytes from A-ob/ob mice demonstrated that Ad-mMTP increased triglyceride secretion without changing the synthesis and secretion of apoB100, suggesting greater incorporation of TG into existing VLDL particles rather than increased particle number. Ad-mMTP administration failed to increase hepatic VLDL secretion in lean Apobec1 2/2 mice or controls. By contrast, VLDL secretion increased and hepatic TG content decreased following Ad-mMTP administration to human APOB transgenic mice crossed into the Apobec1 2/2 line. These findings demonstrate that Ad-mMTP increases murine hepatic VLDL-TG secretion only in the apoB100 background, and even then only in situations with either increased hepatic TG accumulation or increased apoB100 expression. Biosynthesis of hepatic very low density lipoprotein (VLDL) is critically dependent on the coordinated interactions of two dominant proteins, namely apolipoprotein B (apoB) and the microsomal triglyceride transfer protein (Mttp) (as reviewed in Refs. 1-3). ApoB is an obligatory structural component of VLDL and requires progressive lipidation, mediated by the resident endoplasmic reticulum chaperone Mttp, in order to maintain optimal conformational integrity and folding during the process of lipoprotein assembly. In the setting of either limiting Mttp availability or inhibition of Mttp function, the nascent apoB polypeptide becomes misfolded and undergoes presecretory degradation (as reviewed in Refs. 2-4). The importance of these two genes and their interdependent function is illustrated by the phenotypes associated with deletion or mutations in either MTTP and/or APOB gene in humans, which is accompanied by impaired hepatic VLDL secretion and extremely low levels of apoB in plasma (as reviewed in Refs. 3 and 5). These phenotypes have been reproduced using experimentally induced mutations in murine models, suggesting that these integrated and codependent functions are highly conserved (5-7).Studies in rodent hepatoma cells and in primary hepatocytes have revealed an apoB isoform dependence for Mttp-mediated lipidation, with apoB100 exhibiting dramatically more susceptibility to pre...

Abnormalities in very low density lipoprotein (VLDL) assembly and secretion impact intrahepatic lipid homeostasis, plasma lipoprotein profile, and energy metabolism of distal peripheral tissues. We have evaluated the role of the transcriptional coactivator, the peroxisome proliferator-activated receptor-␥ coactivator-1␣ (PGC-1␣), in VLDL assembly and secretion. PGC-1␣ overexpression in HepG2 cells led to diminished rates of triglyceride (TG) synthesis but strongly stimulated VLDL-TG secretion, markedly increasing the efficiency of secretion of newly synthesized TG. PGC-1␣ overexpression increased the rate of secretion of apoB100 and promoted secretion of larger, less dense VLDL particles. PGC-1␣ overexpression in intact mouse liver also stimulated rates of VLDL TG secretion and attenuated hepatic TG accumulation resulting from high fat diet feeding. To determine the molecular mechanisms mediating the effect of PGC-1␣ on VLDL assembly, we evaluated the expression of several candidate mediators known to be involved in VLDL assembly or hepatic lipid homeostasis. Cell death-inducing DFFA-like effector B (CideB) expression was greatly induced by PGC-1␣, and siRNA against CideB reversed the effects of PGC-1␣ on the secretion of TG and VLDL-sized particles by HepG2 cells, indicating that CideB is a critical mediator of stimulatory effects of PGC-1␣ on VLDL secretion. Collectively, these data suggest that PGC-1␣ plays an important role in partitioning cytoplasmic TG toward the VLDL secretory compartments and promoting VLDL secretion via transcriptional induction of CideB. The liver exports triglycerides (TG)2 to extrahepatic tissues through the secretion of very low density lipoproteins (VLDL). Each VLDL particle contains one apolipoprotein B (apoB) molecule, a surface lipid interface composed of phospholipids and free cholesterol, and a hydrophobic core of TG and cholesterol esters (1). Defects in VLDL secretion cause nonalcoholic fatty liver disease in humans (2-4) and mice (5-7). Conversely, overproduction of VLDL is frequently a co-morbidity of obesity, peripheral insulin resistance, and type 2 diabetes mellitus and is a key mechanism leading to the development of dyslipidemia in metabolic syndrome (8 -10). The mechanisms governing VLDL assembly and secretion have yet to be fully understood, and new factors playing important roles in these processes continue to emerge.The biosynthesis of VLDL is generally believed to be a twostep process (11,12). The initial step involves the translocation of the nascent apoB polypeptide across the endoplasmic reticulum (ER) membranes and the cotranslational lipidation of apoB by the microsomal triglyceride transfer protein (MTP). This first step results in the formation of primordial pre-VLDL particles in the high density lipoprotein (HDL)-density range. The second step involves the bulk addition of triglycerides to the VLDL precursors in the smooth ER (13,14) and to a lesser extent in the Golgi apparatus (15, 16), through the fusion of pre-VLDL particles with the luminal lipid dro...

SummaryTo examine interactions between bone morphogenic protein (BMP) and canonical Wnt signaling during skeletal growth, we ablated Smad4, a key component of the TGF-b-BMP pathway, in Osx1 + cells in mice. We show that loss of Smad4 causes stunted growth, spontaneous fractures and a combination of features seen in osteogenesis imperfecta, cleidocranial dysplasia and Wnt-deficiency syndromes. Bones of Smad4 mutant mice exhibited markers of fully differentiated osteoblasts but lacked multiple collagen-processing enzymes, including lysyl oxidase (Lox), a BMP2-responsive gene regulated by Smad4 and Runx2. Accordingly, the collagen matrix in Smad4 mutants was disorganized, but also hypomineralized. Primary osteoblasts from these mutants did not mineralize in vitro in the presence of BMP2 or Wnt3a, and Smad4 mutant mice failed to accrue new bone following systemic inhibition of the Dickkopf homolog Dkk1. Consistent with impaired biological responses to canonical Wnt, ablation of Smad4 causes cleavage of b-catenin and depletion of the low density lipoprotein receptor Lrp5, subsequent to increased caspase-3 activity and apoptosis. In summary, Smad4 regulates maturation of skeletal collagen and osteoblast survival, and is required for matrix-forming responses to both BMP2 and canonical Wnt.

Rationale: Bronchiolitis obliterans syndrome is the leading cause of chronic lung allograft dysfunction. We have demonstrated that respiratory viral infection is a bronchiolitis obliterans syndrome risk factor and virus-dependent injury induces expression of innate airway epithelial genes belonging to the interleukin (IL)-12 family. Thus, we hypothesized that epithelial cell IL-12 family members could mediate lung allograft dysfunction. Objectives: We used mouse and human allograft specimens to evaluate the role of epithelial cell IL-12 family members in allograft dysfunction associated with and without viral infection. Methods: Murine and human IL-12 family members were characterized and manipulated in allografts and then correlated with epithelial cell injury, immune cell accumulation, and collagen deposition. Results: In a mouse model of lung transplantation, concurrent viral infection and allogeneic transplantation increased epithelial injury and this was followed by exaggerated accumulation of macrophages and collagen deposition. This virus-driven allograft dysfunction was associated with an epithelial innate response manifested by a synergistic increase in the production of the macrophage chemoattractant IL-12 p80 (p80), but not IL-12 or IL-23. Blockade or overexpression of donor epithelial p80 resulted in a corresponding abrogation or enhancement of macrophage accumulation and allograft dysfunction. We extended these findings to human recipients with viral infection and transplant bronchitis and again observed excessive epithelial p80 expression that correlated with increased macrophage accumulation. Conclusions: These experiments support a role for an enhanced epithelial innate response as a central process in allograft dysfunction and identify the macrophage chemoattractant p80 as an innate epithelial effector of disease progression.

SummaryCanonical Wnt (cWnt) signaling through b-catenin regulates osteoblast proliferation and differentiation to enhance bone formation. We previously reported that osteogenic action of b-catenin is dependent on BMP signaling. Here, we further examined interactions between cWnt and BMP in bone. In osteoprogenitors stimulated with BMP2, b-catenin localizes to the nucleus, physically interacts with Smad4, and is recruited to DNA-binding transcription complexes containing Smad4, R-Smad1/5 and TCF4. Furthermore, Tcf/Lef-dependent transcription, Ccnd1 expression and proliferation all increase when Smad4, 1 or 5 levels are low, whereas TCF/Lef activities decrease when Smad4 expression is high. The ability of Smad4 to antagonize transcription of Ccnd1 is dependent on DNA-binding activity but Smad4-dependent transcription is not required. In mice, conditional deletion of Smad4 in osterix + cells increases mitosis of cells on trabecular bone surfaces as well as in primary osteoblast cultures from adult bone marrow and neonatal calvaria. By contrast, ablation of Smad4 delays differentiation and matrix mineralization by primary osteoblasts in response to Wnt3a, indicating that loss of Smad4 perturbs the balance between proliferation and differentiation in osteoprogenitors. We propose that Smad4 and Tcf/Lef transcription complexes compete for b-catenin, thus restraining cWnt-dependent proliferative signals while favoring the matrix synthesizing activity of osteoblasts.