Role of Epiregulin on Lipopolysaccharide-Induced Hepatocarcinogenesis as a Mediator via EGFR Signaling in the Cancer Microenvironment.

Lipopolysaccharides (LPSs) have been reported to be important factors in promoting the progression of hepatocellular carcinoma (HCC), but the corresponding molecular mechanisms remain to be elucidated. We hypothesize that epiregulin (EREG), an epidermal growth factor (EGF) family member derived from hepatic stellate cells (HSCs) and activated by LPS stimulation, is a crucial mediator of HCC progression with epidermal growth factor receptor (EGFR) expression in the tumor microenvironment. We used a mouse xenograft model of Huh7 cells mixed with half the number of LX-2 cells, with/without intraperitoneal LPS injection, to elucidate the role of EREG in LPS-induced HCC. In the mouse model, LPS administration significantly enlarged the size of xenografted tumors and elevated the expression of EREG in tumor tissues compared with those in negative controls. Moreover, CD34 immunostaining and the gene expressions of angiogenic markers by a reverse transcription polymerase chain reaction revealed higher vascularization, with increased interleukin-8 (IL-8) expression in the tumors of the mice group treated with LPS compared to those without LPS. Our data collectively suggested that EREG plays an important role in the cancer microenvironment under the influence of LPS to increase not only the tumor cell growth and migration/invasion of EGFR-positive HCC cells but also tumor neovascularization via IL-8 signaling.

ADAMTS-13: A Prognostic Biomarker for Portal Vein Thrombosis in Japanese Patients with Liver Cirrhosis.

Portal vein thrombosis (PVT), one of the most prevalent hepatic vascular conditions in patients with liver cirrhosis (LC), is associated with high mortality rates. An imbalance between a disintegrin-like metalloproteinase with thrombospondin type-1 motifs 13 (ADAMTS-13) enzyme and von Willebrand factor (VWF) is responsible for hypercoagulability, including spontaneous thrombus formation in blood vessels. Herein, we aimed to identify potential prognostic and diagnostic biomarkers in Japanese patients with LC and PVT. In total, 345 patients were divided into two groups: 40 patients who developed PVT (PVT group) and 305 who did not develop PVT (NPVT group). Among the 345 patients with LC, 81% (279/345) were deemed ineligible due to the presence of preventive comorbidities, active or recent malignancies, and organ dysfunction. The remaining 66 patients were divided into two groups: the PVT group (n = 33) and the NPVT group (n = 33). Plasma ADAMTS-13 activity (ADAMTS-13:AC) and the vWF antigen (VWF:Ag) were measured using enzyme-linked immunosorbent assays. Contrast-enhanced, three-dimensional helical computed tomography (CT) was used to detect and characterize PVT. ADAMTS-13:AC was significantly lower in the PVT group than in the NPVT group. No significant differences in plasma vWF:Ag or liver stiffness were observed between the two groups. ADAMTS-13:AC of <18.8 was an independent risk factor for PVT on multivariate analyses (odds ratio: 1.67, 95% confidence interval: 1.21-3.00, p < 0.002). The receiver operating characteristic analysis of ADAMTS-13:AC revealed an area under the curve of 0.913 in PVT detection. Patients with PVT having ADAMTS-13:AC ≥18.8 (n = 17) had higher albumin levels and better prognoses than those with ADAMTS-13:AC <18.8 (n = 16). No significant correlations of ADAMTS-13:AC levels with either fibrin degradation product or D-dimer levels were observed. ADAMTS-13:AC levels could be potential diagnostic and prognostic biomarkers for PVT in Japanese patients with LC.

Changes in Coagulation and Fibrinolytic Factors in Patients With Cirrhotic Refractory Ascites Undergoing Cell-free and Concentrated Ascites Reinfusion Therapy: A Retrospective Observational Study in Japan.

BACKGROUND/AIM: The management of refractory ascites is critical for the treatment of patients with decompensated cirrhosis. This study aimed to evaluate the feasibility and safety of cell-free and concentrated ascites reinfusion therapy (CART) in patients with cirrhosis and refractory ascites, with a focus on changes in coagulation and fibrinolytic factors in ascitic fluid following CART. PATIENTS AND METHODS: This was a retrospective cohort study including 23 patients with refractory ascites undergoing CART. Serum endotoxin activity (EA) before and after CART and the levels of coagulation and fibrinolytic factors and proinflammatory cytokines in original and processed ascitic fluid were measured. The Ascites Symptom Inventory-7 (ASI-7) scale was used for subjective symptom assessment before and after CART. RESULTS: Body weight and waist circumference significantly decreased after CART, whereas serum EA did not significantly change after CART. Similar to the previous reports, ascitic fluid concentrations of total protein, albumin, high-density lipoprotein cholesterol, γ-globulin, and immunoglobulin G levels were significantly increased after CART; mild elevations in body temperature and interleukin 6 and tumor necrosis factor-alpha levels in ascitic fluid were also observed. Importantly, the levels of antithrombin-III, factor VII, and X, which are useful for patients with decompensated cirrhosis, were markedly increased in the reinfused fluid during CART. Finally, the total ASI-7 score was significantly lower following CART, compared with the pre-CART score. CONCLUSION: CART is an effective and safe approach for the treatment of refractory ascites that allows the intravenous reinfusion of coagulation and fibrinolytic factors in the filtered and concentrated ascites.

Sulforaphane Potentiates Gemcitabine-Mediated Anti-Cancer Effects against Intrahepatic Cholangiocarcinoma by Inhibiting HDAC Activity.

Intrahepatic cholangiocarcinoma (iCCA), the second most common primary liver cancer, has high mortality rates because of its limited treatment options and acquired resistance to chemotherapy. Sulforaphane (SFN), a naturally occurring organosulfur compound found in cruciferous vegetables, exhibits multiple therapeutic properties, such as histone deacetylase (HDAC) inhibition and anti-cancer effects. This study assessed the effects of the combination of SFN and gemcitabine (GEM) on human iCCA cell growth. HuCCT-1 and HuH28 cells, representing moderately differentiated and undifferentiated iCCA, respectively, were treated with SFN and/or GEM. SFN concentration dependently reduced total HDAC activity and promoted total histone H3 acetylation in both iCCA cell lines. SFN synergistically augmented the GEM-mediated attenuation of cell viability and proliferation by inducing G2/M cell cycle arrest and apoptosis in both cell lines, as indicated by the cleavage of caspase-3. SFN also inhibited cancer cell invasion and decreased the expression of pro-angiogenic markers (VEGFA, VEGFR2, HIF-1α, and eNOS) in both iCCA cell lines. Notably, SFN effectively inhibited the GEM-mediated induction of epithelial-mesenchymal transition (EMT). A xenograft assay demonstrated that SFN and GEM substantially attenuated human iCCA cell-derived tumor growth with decreased Ki67+ proliferative cells and increased TUNEL+ apoptotic cells. The anti-cancer effects of every single agent were markedly augmented by concomitant use. Consistent with the results of in vitro cell cycle analysis, G2/M arrest was indicated by increased p21 and p-Chk2 expression and decreased p-Cdc25C expression in the tumors of SFN- and GEM-treated mice. Moreover, treatment with SFN inhibited CD34-positive neovascularization with decreased VEGF expression and GEM-induced EMT in iCCA-derived xenografted tumors. In conclusion, these results suggest that combination therapy with SFN with GEM is a potential novel option for iCCA treatment.

Diagnostic yield of liquid-based cytology in serial pancreatic juice aspiration cytological examination.

Objectives: Serial pancreatic juice aspiration cytological examination (SPACE) via endoscopic retrograde cholangiopancreatography is a useful diagnostic method for early-stage pancreatic cancer, such as carcinoma in situ that are difficult to diagnose by endoscopic ultrasound-guided fine needle aspiration (EUS-FNA). However, the diagnostic accuracy of SPACE is low, which is attributed to problems regarding specimen treatment. Hence, we evaluated the diagnostic efficacy of liquid-based cytology (LBC) in pancreatic juice cytology for pancreatic cancer. Methods: We retrospectively analyzed 24 patients with suspected pancreatic cancer that was difficult to diagnose by endoscopic ultrasound-guided fine needle aspiration who underwent SPACE using LBC between April 2017 and April 2021. Results: The most common reason for performing SPACE was localized stenosis of the main pancreatic duct without a mass. Eleven patients were diagnosed with malignancy after surgical resection, nine of whom had pancreatic ductal adenocarcinoma. Ten patients were diagnosed as benign after a follow-up of more than 1 year. The nine cases of malignancy were diagnosed before surgical resection by SPACE using LBC, with a sensitivity of 81.8% and specificity of 100%. The overall diagnostic accuracy was 91.7%. A total of 152 LBC examinations were performed via SPACE, with an adequate sample collection rate of 88.9%. No adverse events, including acute pancreatitis, occurred after endoscopic retrograde cholangiopancreatography. Conclusion: SPACE with LBC offers good diagnostic efficacy in patients with pancreatic cancer that is difficult to diagnose by endoscopic ultrasound-guided fine needle aspiration.

Clinical Significance of Serum Zinc Levels on the Development of Sarcopenia in Cirrhotic Patients.

Background/Aim: Sarcopenia increases the mortality in patients with cirrhosis. Approximately 60% of zinc is accumulated in skeletal muscle. We aimed to determine the role of subclinical zinc deficiency on sarcopenia development in patients with cirrhosis. Patients and Methods: We enrolled 151 patients with cirrhosis and divided them into the group with normal serum zinc levels (Group N: 80-130 µg/dl; n=38) and group with subclinical zinc deficiency (Group D: <80 µg/dl; n=113). The risk factors for sarcopenia were then investigated. Results: Group D had more sarcopenia cases than Group N (31.0% vs. 13.2%). In group D, HGS exhibited a weakly positive but significant correlation with serum zinc levels (R=0.287, p=0.00212), serum zinc levels negatively correlated with both ammonia and myostatin levels (R=-0.254, p=0.0078; R=-0.33, p<0.01), and low zinc levels were independently associated with sarcopenia development. Conclusion: Patients with cirrhosis showing subclinical zinc deficiency have a significantly higher risk of developing sarcopenia.

Enhanced liver fibrosis score as a surrogate of liver-related complications and mortality in primary biliary cholangitis.

ABSTRACT: The presence of bridging fibrosis predicts survival of primary biliary cholangitis (PBC). This study aimed to compare serum parameters for the estimation of liver fibrosis and prediction of clinical outcomes in PBC.Out of 392 patients with PBC, 102 who underwent liver biopsy and in whom fibrosis indices, platelet count, hyaluronic acid, type IV collagen 7 second domain, procollagen type III amino-terminal peptide, tissue inhibitor of metalloproteinases 1, Mac-2 binding protein glycosylation isomer, N-terminal type III collagen propeptide levels; fibrosis index based on 4 factors, aspartate aminotransferase-to-platelet ratio index, and enhanced liver fibrosis (ELF) score were determined, were included. The correlation of histological stages based on both Scheuer and Nakanuma classifications with fibrosis indices was investigated. The Nakanuma system comprises grading for liver fibrosis and bile duct loss. Diagnostic performances of 10 fibrosis indices were evaluated to identify patients with poor prognosis. Moreover, correlations of those with PBC clinical manifestation and survival were also investigated.Enhances liver fibrosis (ELF) score had the highest correlation coefficient for liver fibrosis evaluated according to either the Scheuer or Nakanuma classification among 10 serum fibrosis indices. It also had the highest diagnostic performance in estimating Scheuer stage III and Nakanuma fibrosis score 2, both of which represent portal-bridging fibrosis. Patients with an ELF score of ≥10.0 had shorter survival and presented more frequently clinical complications than those with an ELF score of <10.0.ELF score determines the severity of liver fibrosis and predicts the occurrence of complications and survival in patients with PBC.

Spontaneously ruptured hepatocellular carcinoma in Fontan-associated liver disease: A case report.

The prognosis of congenital heart disease is dramatically improved by cardiac surgery. The Fontan procedure is the definitive palliative operation for patients with single-ventricle physiology. In parallel with the longer survival time achieved with the Fontan procedure, the incidence of Fontan-associated liver disease is increasing. A 40-year-old man who underwent Fontan procedures at the ages of 9 was referred to our hospital for further evaluation of multiple hepatic tumors. Enhanced computed tomography showed large hepatocellular carcinomas with portal thrombi (Vp3). Spontaneous hepatocellular carcinoma rupture occurred 2 weeks after the first visit to our hospital, and emergent transcatheter arterial embolization of the hepatic artery was performed. Three months later, the patient died of liver failure. Autopsy findings showed moderately differentiated hepatocellular carcinoma with a cirrhotic liver characterized by centrilobular fibrosis and sinusoidal dilation similar to that in Fontan-associated liver disease. We reported the first case of spontaneously ruptured hepatocellular carcinoma treated by emergent transcatheter arterial embolization in Fontan-associated liver disease. As the early diagnosis of liver cirrhosis and hepatocellular carcinoma results in better patients' outcome, cardiologists and hepatologists should be aware of Fontan-associated liver disease and advise patients to have regular follow-up of the liver.

Prognostic value of dynamic MRI positive enhancement integral color mapping in osteosynthesis of undisplaced femoral neck fractures.

AIMS: The common treatment for an undisplaced femoral neck fracture is osteosynthesis. Two major complications of osteosynthesis are non-union and late collapse of the femoral head. We speculated that femoral head perfusion is one of the most important factors that affect the outcome of osteosynthesis after femoral neck fracture. We have preoperatively estimated femoral head perfusion by dynamic MRI positive enhancement integral color mapping (PEICM). The purpose of this study was to evaluate the outcomes of undisplaced femoral neck fractures based on PEICM. PATIENTS AND METHODS: Sixty-eight patients participated in this prospective study. All patients underwent PEICM in a 1.5-Tesla MRI machine using coronal fast spoiled gradient echo imaging sequences with gadopentetate dimeglumine as the contrast agent. Femoral head perfusion was displayed via color mapping using PEICM. Three types were distinguished. For type A, the color was identical to unaffected side indicated normal perfusion. For type B, the color was darker than unaffected side indicated decreased perfusion. For type C, the color was black indicated complete absence of perfusion. All patients underwent osteosynthesis with three cannulated screws. The rates of non-union and late collapse for each type were calculated. RESULTS: Sixteen patients were classified as Type A, 43 as Type B, and 6 as Type C. The non-union rates were 0% for Type A, 6.7% for Type B, and 50.0% for Type C. The late collapse rates were 0% for Type A, 4.4% for Type B, and 0% for Type C. CONCLUSION: PEICM precisely detected femoral head perfusion. Primary prosthetic replacement should be considered for older patients with Type C to minimize the chances of revision surgery, even in undisplaced femoral neck fractures.

Myxoid liposarcoma with EWS-CHOP type 1 fusion gene.

BACKGROUND: In myxoid liposarcoma (MLS), the t(12;16)(q13;p11) chromosomal translocation and its resultant fusion transcript, the human translocation liposarcoma (TLS)-CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP), are found in the majority of cases. On the other hand, the variant translocation, t(12;22)(q13;q12) creating the Ewing sarcoma (EWS)-CHOP fusion transcript, is detectable in a limited number of cases. PATIENTS AND METHODS: Tissue from MLS arising in the left thigh of a 19-year-old female was analyzed for possible detection of chromosome translocation and fusion transcript. Fluorescent in situ hybridization (FISH) and reverse transcription-polymerase chain reaction (RT-PCR) methods were used. RESULTS: FISH analysis demonstrated a rearrangement in the CHOP gene. RT-PCR analysis confirmed the presence of EWS-CHOP chimeric transcript type 1, in which exon 7 of EWS was in-frame fused to exon 2 of CHOP with a serine (AGT) to methionine (ATG) transition at the junction. The patient underwent a radical segmental resection including a left vastus medialis musclectomy. Sixty months following the surgical resection, the patient was alive with no evidence of disease. CONCLUSION: Analysis of MLS with EWS-CHOP variant transcripts, type 1 through type 4, including this case together with 15 cases in the literature, indicated that MLS with type 1 fusion transcript may show a more favorable clinical behavior than MLS with other types of fusion transcript.

Specificity of fusion genes in adipocytic tumors.

BACKGROUND: In subsets of adipocytic tumors, specific chromosomal translocations lead to the generation of fusion genes. The high mobility group A2 (HMGA2)-lipoma preferred partner (LPP) and the reciprocal LPP-HMGA2 represent such fusion genes in lipoma, while the human translocation liposarcoma (TLS)-CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP) and the Ewing sarcoma (EWS)-CHOP in liposarcoma. However, the specificity of these fusion genes has not been established in a variety of adipocytic tumors. PATIENTS AND METHODS: One hundred and seventy-two cases of adipocytic tumors, comprising 98 cases of lipoma and 74 cases of liposarcoma, were analyzed for the possible expression of HMGA2-LPP, LPP-HMGA2, TLS-CHOP and EWS-CHOP fusion genes, using a reverse-transcription polymerase chain reaction method. RESULTS: In lipoma, twenty-two cases (22.4%) were associated with either HMGA2-LPP or LPP-HMGA2, while neither TLS-CHOP nor EWS-CHOP transcript was detectable. On the contrary, in liposarcoma, neither HMGA2-LPP nor LPP-HMGA2 transcript was detectable, although twenty-five cases (33.8%) were related to either TLS-CHOP or EWS-CHOP. CONCLUSION: HMGA2-LPP and LPP-HMGA2 were specific to lipoma, and TLS-CHOP and EWS-CHOP were specific to liposarcoma.

Expression of HMGA2-LPP and LPP-HMGA2 fusion genes in lipoma: identification of a novel type of LPP-HMGA2 transcript in four cases.

BACKGROUND: In a subset of lipoma, a specific t(3;12)(q27-28;q14-15) chromosomal translocation leads to the fusion of the high mobility group A2 (HMGA2) gene and the lipoma preferred partner (LPP) gene. Although the expression of HMGA2-LPP fusion gene has been reported in lipomas, the reciprocal LPP-HMGA2 fusion gene has rarely been described. MATERIALS AND METHODS: Ninety-eight cases of lipoma were analyzed for the possible expression of HMGA2-LPP and LPP-HMGA2 fusion genes using a reverse-transcription polymerase chain reaction method. RESULTS: Ten lipomas (10%) revealed both HMGA2-LPP and LPP-HMGA2 fusion transcripts, nine (9%) only HMGA2-LPP, and three (3%) only LPP-HMGA2. DNA sequencing analysis demonstrated that the HMGA2-LPP transcript in 19 lipomas consisted of exons 1-3 of HMGA2 and exons 9-11 of LPP, which was described previously. Out of 13 lipomas with LPP-HMGA2 transcript, 9 were associated with a previously reported LPP-HMGA2 fusion transcript, which fuses exon 8 of LPP to exon 4 of HMGA2, while 4 with a novel type of LPP-HMGA2 fusion transcript, which fuses exon 7 of LPP to exon 4 of HMGA2. CONCLUSION: In addition to the HMGA2-LPP fusion gene, the LPP-HMGA2 fusion gene could have some specific roles for lipomagenesis. The biological implications of the expression and the variation of LPP-HMGA2 fusion transcripts need to be elucidated.

Altered fracture callus formation in chondromodulin-I deficient mice.

Chondromodulin-I (Chm-I) is a glycoprotein that stimulates the growth of chondrocytes and inhibits angiogenesis in vitro. Mice lacking the Chm1 gene show abnormal bone metabolism and pathological angiogenesis in cardiac valves in the mature stage although they develop normally without aberrations in endochondral bone formation during embryogenesis or in cartilage development during growth. These findings indicate that Chm-I is critical under conditions of stress such as bone repair through endochondral ossification of a fracture callus. We carried out the present study to examine the expression and role of Chm-I in bone repair using a stabilized tibial fracture model, and compared fracture healing in Chm1 knockout (Chm1(-/-)) mice with that in wild-type mice. Chm-I mRNA and protein localized in the external cartilaginous callus in the reparative phase of fracture healing. Radiological examination showed a delayed union in Chm1(-/-) mice although the fracture site was covered with both external and internal calluses. Chm1 null mutation reduced external cartilaginous callus formation as judged by marked decrease of type X collagen alpha 1 (Col10a1) expression and the total amount of cartilage matrix. Interestingly, the majority of chondrocytes in the periosteal callus failed to differentiate into mature chondrocytes in Chm1(-/-) mice, while the hypertrophic maturation of chondrocytes between the cortices was not affected. These results suggest that Chm-I is involved in hypertrophic maturation of periosteal chondrocytes. Although a direct effect of Chm-I on bones is still unclear, bony callus formation was increased while external cartilaginous callus decreased in Chm1(-/-) mice. We conclude that in the absence of Chm1, predominant primary bone healing occurs due to an indirect effect induced by reduction of cartilaginous callus rather than to a direct effect on osteogenic function, resulting in a delayed union.

A novel type of EWS-CHOP fusion gene in myxoid liposarcoma.

The cytogenetic hallmark of myxoid type and round cell type liposarcoma consists of reciprocal translocation of t(12;16)(q13;p11) and t(12;22)(q13;q12), which results in fusion of TLS/FUS and CHOP, and EWS and CHOP, respectively. Nine structural variations of the TLS/FUS-CHOP chimeric transcript have been reported, however, only two types of EWS-CHOP have been described. We describe here a case of myxoid liposarcoma containing a novel EWS-CHOP chimeric transcript and identified the breakpoint occurring in intron 13 of EWS. Reverse transcription-polymerase chain reaction and direct sequence showed that exon 13 of EWS was in-frame fused to exon 2 of CHOP. Genomic analysis revealed that the breaks were located in intron 13 of EWS and intron 1 of CHOP.

MRI characteristics of parosteal lipomas associated with the HMGA2-LPP fusion gene.

BACKGROUND: The magnetic resonance (MR) characteristics of parosteal lipomas with the HMGA2-LPP fusion transcripts are described. PATIENTS AND METHODS: The expression of HMGA2-LPP fusion transcripts was determined using the reverse transcription-polymerase chain reaction method. RESULTS: MR images of two cases with the fusion transcripts, a 56-year-old man and a 50-year-old woman, revealed heterogeneous high signal intensities on T1- and T2-weighted images, showing heterogeneous curvilinear enhancement on fat-suppressed T1-weighted images after Gd-DTPA injection, which resembled those of well-differentiated liposarcomas. CONCLUSION: Since the HMGA2-LPP fusion transcripts are exclusively detectable in benign mesenchymal tumors, testing HMGA2-LPP expression may be useful for differential diagnosis in cases of radiologically-suspected well-differentiated liposarcomas.

Sp1 family of transcription factors regulates the human alpha2 (XI) collagen gene (COL11A2) in Saos-2 osteoblastic cells.

UNLABELLED: Genes encoding type XI collagen, normally associated with chondrogenesis, are also expressed by osteoblasts. By studying Saos-2 cells, we showed that the transcription factors, Sp1, Sp3, and Sp7 (Osterix), regulate COL11A2 expression through its proximal promoter. The findings indicate both ubiquitous and osteoblast-specific mechanisms of collagen gene regulation. INTRODUCTION: Type XI collagen is essential for skeletal morphogenesis. Collagen XI gene regulation has been studied in chondrocytes but not in osteoblasts. MATERIALS AND METHODS: We cultured Saos-2 cells, a human osteosarcoma-derived line of osteoblasts, and analyzed them for alpha2(XI) protein and COL11A2 regulatory mechanisms. RESULTS AND CONCLUSIONS: Although types I and V were the dominant collagens deposited by Saos-2 cells, they expressed COL11A2 mRNA, and alpha2(XI) chains were present in the extracellular matrix. The COL11A2 promoter region (from -149 to -40) containing three Sp1 binding sites was required for promoter activity in transient transfection assays. All three Sp1 sites were critical for binding by nuclear proteins in electrophoretic mobility shift assays. Further analysis using consensus oligonucleotides and specific antibodies as well as chromatin immunoprecipitation assay implicated Sp1 and Sp3 in binding to this promoter region. Overexpressing Sp1 or Sp3 significantly increased COL11A2 promoter activity and endogenous COL11A2 gene expression, an effect that was suppressed by the Sp1-binding inhibitor mithramycin A. Further experiments showed that Sp1, Sp3, CREB-binding protein (CBP), p300, and histone deacetylase (HDAC) were physically associated and HDAC inhibitors (trichostatin A or NaB) upregulated COL11A2 promoter activity and endogenous gene expression. Another Sp1 family member, Sp7 (Osterix), was expressed in Saos-2 cells, but not in chondrocytes, and was shown by chromatin immunoprecipitation to occupy the COL11A2 promoter. Overexpressing Sp7 increased COL11A2 promoter activity and endogenous gene expression, an effect also blocked by mithramycin A. Using siRNA to knockdown Sp1, Sp3, or Sp7, it was shown that depression of any of them decreased COL11A2 promoter activity and endogenous gene expression. Finally, primary cultures of osteoblasts expressed COL11A2 and Sp7, upregulated COL11A2 promoter activity and endogenous gene expression when Sp1, Sp3, or Sp7 were overexpressed, and downregulated them when Sp1, Sp3, or Sp7 were selectively depressed. The results establish that Sp1 proteins regulate COL11A2 transcription by binding to its proximal promoter and directly interacting with CBP, p300, and HDAC.

Overexpression of HMGA2-LPP fusion transcripts promotes expression of the alpha 2 type XI collagen gene.

In a subset of human lipomas, a specific t(3;12) chromosome translocation gives rise to HMGA2-LPP fusion protein, containing the amino (N)-terminal DNA binding domains of HMGA2 fused to the carboxyl (C)-terminal LIM domains of LPP. In addition to its role in adipogenesis, several observations suggest that HMGA2-LPP is linked to chondrogenesis. Here, we analyzed whether HMGA2-LPP promotes chondrogenic differentiation, a marker of which is transactivation of the alpha 2 type XI collagen gene (Col11a2). Real-time PCR analysis showed that HMGA2-LPP and COL11A2 were co-expressed. Luciferase assay demonstrated that either of HMGA2-LPP, wild-type HMGA2 or the N-terminal HMGA2 transactivated the Col11a2 promoter in HeLa cells, while the C-terminal LPP did not. RT-PCR analysis revealed that HMGA2-LPP transcripts in lipomas with the fusion were 591-fold of full-length HMGA2 transcripts in lipomas without the fusion. These results indicate that in vivo overexpression of HMGA2-LPP promotes chondrogenesis by upregulating cartilage-specific collagen gene expression through the N-terminal DNA binding domains.

Differential expression of VEGF isoforms and VEGF receptors in cartilaginous tumors.

Vascular endothelial growth factor (VEGF), which is known to have at least five isoforms and four receptors, is integral to tumor-induced neovascularization. In order to determine whether VEGF signaling is modulated in the development of cartilaginous tumors, we analyzed osteochondrogenic tumors for the expression of VEGF isoforms and VEGF receptors using the reverse transcription-polymerase chain reaction method. Although mRNA for VEGF121, VEGF165, VEGFR-1 and NRP-2 was widely expressed, mRNA expression for VEGF189, VEGFR-2 and NRP-1 was linked to a malignant phenotype such as local invasion. These results indicated that VEGF signaling was fine-tuned by the differential expression of the VEGF isoforms and VEGF receptors.