Cbfa1 is a transcription factor that belongs to the runt domain gene family. Cbfa1-deficient mice showed a complete lack of bone formation due to the maturational arrest of osteoblasts, demonstrating that Cbfa1 is an essential factor for osteoblast differentiation. Further, chondrocyte maturation was severely disturbed in Cbfa1-deficient mice. In this study, we examined the possibility that Cbfa1 is also involved in the regulation of chondrocyte differentiation. mRNAs for both Cbfa1 isotypes, type I Cbfa1 (Pebp2␣A/Cbfa1) and type II Cbfa1 (Osf2/Cbfa1 or til-1), which are different in N-terminal domain, were expressed in terminal hypertrophic chondrocytes as well as osteoblasts. In addition, mRNA for type I Cbfa1 was expressed in other hypertrophic chondrocytes and prehypertrophic chondropcytes. In a chondrogenic cell line, ATDC5, the expression of type I Cbfa1 was elevated prior to differentiation to the hypertrophic phenotype, which is characterized by type X collagen expression. Treatment with antisense oligonucleotides for type I Cbfa1 severely reduced type X collagen expression in ATDC5 cells. Retrovirally forced expression of either type I or type II Cbfa1 in chick immature chondrocytes induced type X collagen and MMP13 expression, alkaline phosphatase activity, and extensive cartilage-matrix mineralization. These results indicate that Cbfa1 is an important regulatory factor in chondrocyte maturation.
During skeletogenesis, cartilage develops to either permanent cartilage that persists through life or transient cartilage that is eventually replaced by bone. However, the mechanism by which cartilage phenotype is specified remains unclarified. Core binding factor α1 (Cbfa1) is an essential transcription factor for osteoblast differentiation and bone formation and has the ability to stimulate chondrocyte maturation in vitro. To understand the roles of Cbfa1 in chondrocytes during skeletal development, we generated transgenic mice that overexpress Cbfa1 or a dominant negative (DN)-Cbfa1 in chondrocytes under the control of a type II collagen promoter/enhancer. Both types of transgenic mice displayed dwarfism and skeletal malformations, which, however, resulted from opposite cellular phenotypes. Cbfa1 overexpression caused acceleration of endochondral ossification due to precocious chondrocyte maturation, whereas overexpression of DN-Cbfa1 suppressed maturation and delayed endochondral ossification. In addition, Cbfa1 transgenic mice failed to form most of their joints and permanent cartilage entered the endochondral pathway, whereas most chondrocytes in DN-Cbfa1 transgenic mice retained a marker for permanent cartilage. These data show that temporally and spatially regulated expression of Cbfa1 in chondrocytes is required for skeletogenesis, including formation of joints, permanent cartilages, and endochondral bones.
BackgroundWe aimed to elucidate the relationship between serum myostatin levels and other markers including skeletal muscle mass and to investigate the influence of serum myostatin levels on survival for patients with liver cirrhosis (LC).MethodsA total of 198 LC subjects were analysed in this study. Myostatin levels were measured using stored sera. We retrospectively investigated the relationship between myostatin level and other markers, and the influence of myostatin level on overall survival (OS). Assessment of skeletal muscle mass was performed using the psoas muscle index (PMI) on computed tomography images at baseline. PMI indicates the sum of bilateral psoas muscle mass calculated by hand tracing at the lumber three level on computed tomography images divided by height squared (cm2/m2). The study cohort was divided into two groups based on the median myostatin value in each gender.ResultsOur study cohort included 108 male and 90 female patients with a median age of 67.5 years. The median (range) myostatin level for male patients was 3419.6 pg/mL (578.4–12897.7 pg/mL), whereas that for female patients was 2662.4 pg/mL (710.4–8782.0 pg/mL) (P = 0.0024). Median (range) serum myostatin level for Child–Pugh A patients (n = 123) was 2726.0 pg/mL (578.4–12667.2 pg/mL), whereas that for Child–Pugh B or C patients (n = 75) was 3615.2 pg/mL (663.3–12897.7 pg/mL) (P = 0.0011). For the entire cohort, the 1‐, 3‐, 5‐, and 7‐year cumulative OS rates were 93.94%, 72.71%, 50.37%, and 38.47%, respectively, in the high‐myostatin group and 96.97%, 83.27%, 73.60%, and 69.95%, respectively, in the low‐myostatin group (P = 0.0001). After excluding hepatocellular carcinoma patients (at baseline) from our analysis (n = 158), the 1‐, 3‐, 5‐, and 7‐year cumulative OS rates were 96.0%, 77.93%, 52.97%, and 39.08%, respectively, in the high‐myostatin group and 96.39%, 87.58%, 77.63%, and 73.24%, respectively, in the low‐myostatin group (P = 0.0005). Higher age (P = 0.0111) and lower PMI (P < 0.0001) were identified as significant predictors of poorer OS in our multivariate analysis, while higher serum myostatin (P = 0.0855) tended to be a significant adverse predictor. In both genders, PMI, serum albumin, prothrombin time, and branched‐chain amino acid to tyrosine ratio showed a significantly inverse correlation with myostatin levels, and serum ammonia levels showed a significantly positive correlation with myostatin levels.ConclusionsHigher serum myostatin levels correlated with muscle mass loss, hyperammonemia, and impaired protein synthesis, as reflected by lower serum albumin levels and lower branched‐chain amino acid to tyrosine ratio levels. High serum myostatin levels were also associated with a reduced OS rate in LC patients.
Hepatoma-derived growth factor (HDGF) is the original member of the HDGF family of proteins, which contains a well-conserved N-terminal amino acid sequence (homologous to the amino terminus of HDGF; hath) and nuclear localization signals (NLSs) in gene-specific regions other than the hath region. In addition to a bipartite NLS in a gene-specific region, an NLS-like sequence is also found in the hath region. In cells expressing green fluorescence protein (GFP)-HDGF, green fluorescence was observed in the nucleus, whereas it was detected in the cytoplasm of cells expressing GFP-HDGF with both NLSs mutated or deleted. GFP-hath protein (GFP-HATH) was distributed mainly in the nucleus, although some was present in the cytoplasm, whereas GFP-HDGF with a deleted hath region (HDGFnonHATH) was found only in the nucleus. Exogenously supplied GFP-HDGF was internalized and translocated to the nucleus. GFP-HATH was internalized, whereas GFP-HDGFnonHATH was not. Overexpression of HDGF stimulated DNA synthesis and cellular proliferation, although HDGF with both NLSs deleted did not. Overexpression of HDGFnonHATH caused a significant stimulation of DNA synthesis, whereas that of hath protein did not. HDGF containing the NLS sequence of p53 instead of the bipartite NLS did not stimulate DNA synthesis, and truncated forms without the C- or N-terminal side of NLS2 did not. These findings suggest that the gene-specific region, at least the bipartite NLS sequence and the N- and C-terminal neighboring portions, is essential for the mitogenic activity of HDGF after nuclear translocation.
Receptor activator of nuclear factor-B ligand (RANKL), osteoprotegerin (OPG), and macrophage-colony stimulating factor play essential roles in the regulation of osteoclastogenesis. Runx2-deficient (Runx2 ؊/؊ ) mice showed a complete lack of bone formation because of maturational arrest of osteoblasts and disturbed chondrocyte maturation. Further, osteoclasts were absent in these mice, in which OPG and macrophage-colony stimulating factor were normally expressed, but RANKL expression was severely diminished. We investigated the function of Runx2 in osteoclast differentiation. A Runx2 ؊/؊ calvaria-derived cell line (CA120 -4), which expressed OPG strongly but RANKL barely, severely suppressed osteoclast differentiation from normal bone marrow cells in co-cultures. Adenoviral introduction of Runx2 into CA120 -4 cells induced RANKL expression, suppressed OPG expression, and restored osteoclast differentiation from normal bone marrow cells, whereas the addition of OPG abolished the osteoclast differentiation induced by Runx2. Addition of soluble RANKL (sRANKL) also restored osteoclast differentiation in co-cultures. Forced expression of sRANKL in Runx2 ؊/؊ livers increased the number and size of osteoclast-like cells around calcified cartilage, although vascular invasion into the cartilage was superficial because of incomplete osteoclast differentiation. These findings indicate that Runx2 promotes osteoclast differentiation by inducing RANKL and inhibiting OPG. As the introduction of sRANKL was insufficient for osteoclast differentiation in Runx2 ؊/؊ mice, however, our findings also suggest that additional factor(s) or matrix protein(s), which are induced in terminally differentiated chondrocytes or osteoblasts by Runx2, are required for osteoclastogenesis in early skeletal development.In the process of endochondral ossification, chondrocytes mature to hypertrophic chondrocytes, matrix around terminally differentiated chondrocytes (terminal hypertrophic chondrocytes) is mineralized, blood vessels invade into the calcified cartilage, and cartilage is replaced by bone (1). Osteoclasts accelerate these processes by resorption of the calcified matrix leading to bone marrow formation. Osteoclasts differentiate from hematopoietic precursor cells through direct contact with osteoblastic/stromal cells (2). Recently, osteoprotegerin (OPG) 1 / osteoclastogenesis inhibitory factor, which is an inhibitor of osteoclast differentiation (3, 4), and receptor activator of NF-B (RANK) ligand (RANKL)/tumor necrosis factor-related activation-induced cytokine/OPG ligand/osteoclast differentiation factor, which is an inducer of osteoclast differentiation (5-8), were identified. RANKL, which is expressed on the surface of osteoblastic/stromal cells or released as a soluble factor, binds to its receptor RANK (9, 10), which is expressed on the surface of osteoclast precursors and osteoclasts, and induces osteoclast differentiation and activation. OPG, which binds RANKL with higher affinity than RANK, acts as a decoy receptor for RANKL and in...
Runx2 (runt-related transcription factor 2) is an important transcription factor for chondrocyte differentiation as well as for osteoblast differentiation. To investigate the function of Runx2 in chondrocytes, we isolated chondrocytes from the rib cartilage of Runx2-deficient (Runx2–/–) mice and examined the effect of Runx2 deficiency on chondrocyte function and behavior in culture for up to 12 days. At the beginning of the culture, Runx2–/– chondrocytes actively proliferated, had a polygonal shape and expressed type II collagen; these are all characteristics of chondrocytes. However, they gradually accumulated lipid droplets that stained with oil red O and resembled adipocytes. Northern blot analysis revealed that the expression of adipocyte-related differentiation marker genes including PPARγ (peroxisome proliferator-activated receptor γ), aP2 and Glut4 increased over time in culture, whereas expression of type II collagen decreased. Furthermore, the expression of Pref-1, an important inhibitory gene of adipogenesis, was remarkably decreased. Adenoviral introduction of Runx2 or treatment with transforming growth factor-β, retinoic acid, interleukin-1β, basic fibroblast growth factor, platelet-derived growth factor or parathyroid hormone inhibited the adipogenic changes in Runx2–/– chondrocytes. Runx2 and transforming growth factor-β synergistically upregulated interleukin-11 expression, and the addition of interleukin-11 to the culture medium reduced adipogenesis in Runx2–/– chondrocytes. These findings indicate that depletion of Runx2 resulted in the loss of the differentiated phenotype in chondrocytes and induced adipogenic differentiation in vitro, and show that Runx2 plays important roles in maintaining the chondrocyte phenotype and in inhibiting adipogenesis. Our findings suggest that these Runx2-dependent functions are mediated, at least in part, by interleukin-11.
Background To assess the recent real-world changes in the etiologies of liver cirrhosis (LC) in Japan, we conducted a nationwide survey in the annual meeting of the Japan Society of Hepatology (JSH). Methods We investigated the etiologies of LC patients accumulated from 68 participants in 79 institutions (N = 48,621). We next assessed changing trends in the etiologies of LC by analyzing cases in which the year of diagnosis was available (N = 45,834). We further evaluated the transition in the real number of newly identified LC patients by assessing data from 36 hospitals with complete datasets for 2008-2016 (N = 18,358). Results In the overall data, HCV infection (48.2%) was the leading cause of LC in Japan, and HBV infection (11.5%) was the third-most common cause. Regarding the transition in the etiologies of LC, the contribution of viral hepatitisrelated LC dropped from 73.4 to 49.7%. Among the nonviral etiologies, alcoholic-related disease (ALD) and nonalcoholic steatohepatitis (NASH)-related LC showed a notable increase (from 13.7 to 24.9% and from 2.0 to 9.1%, respectively). Regarding the real numbers of newly diagnosed patients from 2008 to 2016, the numbers of patients with viral hepatitis-related LC decreased, while the numbers of patients with non-viral LC increased.Conclusions HCV has remained the main cause of LC in Japan; however, the contribution of viral hepatitis as an etiology of LC is suggested to have been decreasing. In addition, non-viral LC, such as ALD-related LC and NASH-related LC, is suggested to have increased as etiologies of LC in Japan.
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