Hirayuki Enomoto

Runx2 deficiency in chondrocytes causes adipogenic changes in vitro

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.

Hepatoma-derived Growth Factor Stimulates Cell Growth after Translocation to the Nucleus by Nuclear Localization Signals

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.

Hepatoma-Derived Growth Factor Is a Novel Prognostic Factor for Patients with Pancreatic Cancer

Purpose: Hepatoma-derived growth factor (HDGF) is a nucleus-targeted growth factor playing an important role in the development and progression of cancers. This study investigated the correlation of HDGF expression and prognosis in patients with pancreatic ductal carcinoma. Patients and Methods: HDGF expression in pancreatic cancer cell lines was analyzed by Western blotting. HDGF expression was analyzed by immunohistochemistry for 50 patients with primary ductal carcinoma of the pancreas (33 male and 17 female) ranging in age from 48 to 80 years (median, 65 years) receiving surgical treatment. Cancer cells showing stronger staining than the noncancerous ducts were regarded as positive. Cases showing positive staining in <90% and >90% of tumor cells were regarded as HDGF labeling index (LI) levels 1 and 2, respectively. HDGF LI was determined separately for the nucleus and the cytoplasm. Results: Western blotting showed HDGF expression in pancreatic cancer cells similar to that of hepatic cell lines. Twenty-three (46%) and 27 (54%) cases and 22 (44%) and 28 (56%) cases showed HDGF LI levels 1 and 2 for the nucleus and the cytoplasm, respectively. Patients with nuclear HDGF LI level 1 showed a significantly better 5-year survival rate (37.0%) than those with level 2 (6.8%; P = 0.023). No significant difference was observed in the cytoplasmic HDGF LI classification. Multivariate analysis revealed nuclear HDGF LI to be an independent prognosticator. Conclusions: These findings suggest that HDGF could be a novel prognostic factor for pancreatic ductal carcinoma.

Hepatoma‐derived growth factor is highly expressed in developing liver and promotes fetal hepatocyte proliferation

Hepatoma‐derived growth factor (HDGF) is a heparin‐binding protein, which has been purified from the conditioned media of HuH‐7 hepatoma cells. Recent studies have suggested the involvement of HDGF in development of the kidney and cardiovascular systems. In the present study, we investigated the possibility that HDGF was also involved in liver development. Northern blot and immunostaining revealed unique expression patterns of HDGF in liver development. HDGF expression was strongly detected in the fetal liver of the midgestation stage and was markedly decreased near birth. Its expression was mainly detected in stromal cells, including immature hepatocytes. Expression in hepatocytes decreased with differentiation. Administration of recombinant HDGF enhanced the growth of primary cultured fetal hepatocytes significantly, although the effect was small. The effect of exogenous HDGF on the proliferation of neonatal hepatocytes was also small and significant only at one point, despite the lower expression of endogenous HDGF, suggesting that the differences exist between fetal and neonatal hepatocytes. However, adenoviral introduction of HDGF antisense cDNA into the fetal hepatocytes significantly suppressed their proliferation, and the inhibitory effect of HDGF antisense virus was reversed by exogenous HDGF. In conclusion, HDGF helps regulate the hepatocyte proliferation in liver development. (HEPATOLOGY2002;36:1519–1527).

Hepatoma-derived growth factor induces tumorigenesis in vivo through both direct angiogenic activity and induction of vascular endothelial growth factor

Hepatoma‐derived growth factor (HDGF) is highly expressed in tumor cells, and stimulates their proliferation. In the present study, we investigated the role of HDGF in tumorigenesis and elucidated the mechanism of action. Stable transfectants of NIH3T3 cells overexpressing HDGF did not show significant anchorage‐independent growth in soft agar assay. However, these stable transfectants overexpressing HDGF generated sarcomatous tumors in nude mice. These tumors were red‐colored macroscopically, and histologically showed a rich vascularity. Immunohistochemical analysis using CD31 antibody showed new vessel formation. Recombinant HDGF stimulated proliferation of human umbilical vein endothelial cells in a dose‐dependent manner, and stimulated tubule formation. Furthermore, vascular endothelial growth factor (VEGF) was detected immunohistochemically in the tumor tissues. Transient expression of HDGF induced both VEGF gene and protein expression as demonstrated by a reporter assay using VEGF gene promoter. The administration of anti‐VEGF neutralizing antibody significantly suppressed, but did not block, the tumor growth of HDGF‐overexpressing cells in nude mice. Thus, these findings suggested that HDGF‐induced tumor formation in vivo involves induction of VEGF as well as direct angiogenic activity.

Hepatoma-Derived Growth Factor Is a Novel Prognostic Factor for Hepatocellular Carcinoma

BackgroundHepatoma-derived growth factor (HDGF) is involved in hepatocarcinogenesis, as well as in liver development and regeneration. This study investigated the correlation of HDGF expression with differentiation and prognosis of hepatocellular carcinoma (HCC).MethodsHDGF expression in 100 patients with HCC (81 men and 19 women) with ages ranging from 34 to 81 years (median, 61 years) receiving surgical treatment was analyzed by immunohistochemistry. HDGF messenger RNA expression was evaluated in 10 cases by reverse transcription-polymerase chain reaction. The immunostaining pattern in HCCs was categorized as a positive HDGF index (showing positive staining in >90% of tumor cells in both nucleus and cytoplasm) or a negative HDGF index (all others).ResultsTwenty-seven cases (27%) showed a positive and 73 (73%) showed a negative HDGF index. HDGF messenger RNA expression was significantly higher in four cases with a positive HDGF index than in six with a negative index. Cases with well-differentiated histological characteristics showed a higher rate of positive HDGF index than those with a poorly differentiated subtype. Univariate and multivariate analysis revealed significantly poorer disease-free and overall survivals in patients with a positive HDGF index compared with patients with a negative index.ConclusionsThese findings suggest the potential utility of HDGF immunohistochemistry in determining the prognosis of HCC.

Expression of hepatoma-derived growth factor in hepatocarcinogenesis

Background and Aim:  The present study investigated the expression of hepatoma‐derived growth factor (HDGF) in human hepatocellular carcinoma (HCC) and in the liver during hepatocarcinogenesis in two rodent models.

Impaired vascular invasion of Cbfa1-deficient cartilage engrafted in the spleen.

Chondrocyte maturation and vascular invasion of cartilage are essential in the process of endochondral ossification. Cbfa1‐deficient (Cbfa1−/−) mice displayed a complete absence of osteoblast and osteoclast maturation as well as severely inhibited chondrocyte maturation in most parts of the skeleton. Although chondrocyte maturation and mineralization were observed in restricted areas of Cbfa1−/− mouse skeleton, vascular invasion of calcified cartilage was never noted. To investigate the possibility of chondrocyte maturation and vascular invasion in Cbfa1−/− cartilage and the role of the hematopoietic system in the process of vascular invasion, we transplanted embryonic day 18.5 (E18.5) Cbfa1−/− femurs, which are composed of immature chondrocytes, into spleens of normal mice. One week later, the transplanted femurs contained terminally differentiated chondrocytes expressing osteopontin, bone sialoprotein (BSP), and matrix metalloproteinase (MMP) 13. In the diaphyses of the transplants, the cartilage matrix was mineralized and the cartilage was invaded by vascular vessels and osteoclasts. However, chondrocyte maturation and vascular invasion were severely retarded in comparison with transplants of E14.5 wild‐type femurs, in which the cartilage was rapidly replaced by bone, and neither mature osteoblasts nor bone formation were observed. In primary culture of Cbfa1−/− chondrocytes, transforming growth factor (TGF) β1, platelet‐derived growth factor (PDGF), interleukin (IL)‐1β, and thyroid hormone (T3) induced osteopontin and MMP‐13 expression. These findings indicated that factors in the hematopoietic system are able to support vascular invasion of cartilage independent of Cbfa1 but are less effective without it, suggesting that Cbfa1 functions in cooperation with factors from bone marrow in the process of growth plate vascularization.

Participation of hepatoma-derived growth factor in the regulation of fetal hepatocyte proliferation

The identification and characterization of hepatic stem cell compartments is the key to resolving clinical disorders and diseases in the liver. Hepatoblasts (or early fetal hepatocytes) fulfill several criteria of hepatic stem cells during liver development. Unlike mature hepatocytes, immature fetal hepatocytes can proliferate autonomously in the absence of any growth factors in vitro. However, the regulation of fetal hepatocyte proliferation remains unclear. Recently, we identified a novel factor, hepatoma-derived growth factor (HDGF), from the human hepatoma-derived cell line HuH-7, which autonomously proliferate in serum-free defined medium. Here, we focus on the functional roles of HDGF, and review several molecules involved in the growth regulation of hepatocytes in the immature stage.

Forced expression of terminal deoxynucleotidyl transferase in fetal thymus resulted in a decrease in γδ T cells and random dissemination of Vγ3Vδ1 T cells in skin of newborn but not adult mice

The repertoire of lymphocyte receptor genes encoded in a germline is further diversified by a number of processes, including the template‐independent addition of nucleotides (N regions) by means of terminal deoxynucleotidyl transferase (TdT). Normally, mouse γδ T cells in the early fetal thymus, whose T‐cell receptor (TCR) genes lack N regions and are encoded by Vγ3‐Jγ1 and Vδ1‐Dδ2‐Jδ2 with canonical junctions (invariant Vγ3Vδ1), are thought to be the precursors of dendritic epidermal T cells (DETC). We generated mutant mice whose endogenous TdT promoter was replaced with the lck promoter through homologous recombination. These mutant mice expressed TdT in fetal thymus, had abundant N regions and infrequent canonical junctions in γ and δ rearrangements, and showed a decreased number of γδ T cells. Various Vγ3Vδ1 T cells, most of which had N regions in their TCR genes, were found to disseminate in the skin of newborn mutant mice, whereas normal numbers of DETCs with the invariant Vγ3Vδ1 rearrangement were observed in adult mutants. These data demonstrate that the regulation of TdT expression during fetal development is important for the generation of γδ T cells, and that Vγ3Vδ1 T cells, which have various junctional sequences in their TCR genes, randomly disseminate in skin, but invariant Vγ3Vδ1 T cells have a great advantage for proliferation in skin.