Protein HMG-I Research Articles

Retraction for Melillo et al., "Critical Role of the HMGI(Y) Proteins in Adipocytic Cell Growth and Differentiation".

Retraction for Melillo et al., "Critical Role of the HMGI(Y) Proteins in Adipocytic Cell Growth and Differentiation".

LRP6 Enhances Glucose Metabolism by Promoting TCF7L2-Dependent Insulin Receptor Expression and IGF Receptor Stabilization in Humans

Common genetic variations in Wnt signaling genes have been associated with metabolic syndrome and diabetes by mechanisms that are poorly understood. A rare nonconservative mutation in Wnt coreceptor LRP6 (LRP6(R611C)) has been shown to underlie autosomal dominant early onset coronary artery disease, type 2 diabetes, and metabolic syndrome. We examined the interplay between Wnt and insulin signaling pathways in skeletal muscle and skin fibroblasts of healthy nondiabetic LRP6(R611C) mutation carriers. LRP6 mutation carriers exhibited hyperinsulinemia and reduced insulin sensitivity compared to noncarrier relatives in response to oral glucose ingestion, which correlated with a significant decline in tissue expression of the insulin receptor and insulin signaling activity. Further investigations showed that the LRP6(R611C) mutation diminishes TCF7L2-dependent transcription of the IR while it increases the stability of IGFR and enhances mTORC1 activity. These findings identify the Wnt/LRP6/TCF7L2 axis as a regulator of glucose metabolism and a potential therapeutic target for insulin resistance.

Repression of Smooth Muscle Differentiation by a Novel High Mobility Group Box-containing Protein, HMG2L1

The molecular mechanisms regulating smooth muscle-specific gene expression during smooth muscle development are poorly understood. Myocardin is an extraordinarily powerful cofactor of serum response factor (SRF) that stimulates expression of smooth muscle-specific genes. In an effort to search for proteins that regulate myocardin function, we identified a novel HMG box-containing protein HMG2L1 (high mobility group 2 like 1). We found that HMG2L1 expression is correlated with the smooth muscle cell (SMC) synthetic phenotype. Overexpression of HMG2L1 in SMCs down-regulated smooth muscle marker expression. Conversely, depletion of endogenous HMG2L1 in SMCs increases smooth muscle-specific gene expression. Furthermore, we found HMG2L1 specifically abrogates myocardin-induced activation of smooth muscle-specific genes. By GST pulldown assays, the interaction domains between HMG2L1 and myocardin were mapped to the N termini of each of the proteins. Finally, we demonstrated that HMG2L1 abrogates myocardin function through disrupting its binding to SRF and abolishing SRF-myocardin complex binding to the promoters of smooth muscle-specific genes. This study provides the first evidence of this novel HMG2L1 molecule playing an important role in attenuating smooth muscle differentiation.

High mobility group HMGI(Y) protein expression in head and neck squamous cell carcinoma

Conclusion. We conclude that increased expression level of the high mobility group I (HMGI(Y)) is closely associated with malignant transformation in head and neck squamous cell carcinomas (HNSCCs), and the measurement of HMGI(Y) levels in HNSCCs may be useful as a prognostic marker. Objectives. To investigate whether HMGI overexpression is observed in HNSCCs, and its value as a prognostic marker in HNSCCs. Materials and methods. HMGI(Y) expression was determined at the protein level by immunohistochemisty using a HMGI(Y)-specific antibody and RT-PCR in 10 surgically resected specimens of non-neoplastic tissue (normal palatal tissue) and 40 HNSCCs. We also evaluated the association of HMGI(Y) overexpression within clinicopathologic parameters, i.e. clinical stage, pathologic grade, status of cervical lymph node metastasis, recurrence rate. Results. Expression of HMGI(Y) by immunohistochemical staining was observed in 35 of 40 (87.5%) HNSCC samples, whereas normal mucosa and/or the mucosa adjacent to the tumor tissue showed negative or weakly positive staining (p<0.05). Semi-quantification of HMGI(Y) by RT-PCR was 2.98±2.24 in cancer and 0.47±0.25 in normal tissue (p<0.001). High expression of HMGI(Y) was observed in recurrent cases, compared with non-recurrent cases (p<0.05). However, no significant correlation was observed between the levels of HMGI(Y) expression and other clinical factors such as clinical stage, pathologic grade, and status of cervical lymph node metastasis.

Expression of the High-Mobility Group Protein HMGI(Y) in Gestational Trophoblastic Diseases

The high-mobility group protein HMGI(Y) is a member of a family of nonhistone chromosomal proteins, which have been implicated in the regulation of inducible gene transcription, integration of retroviruses into chromosomes, and induction of neoplastic transformation and metastatic progression in cancer cells. The human trophoblast is a tissue that shares proliferation capacity and invasiveness with neoplastic tissues, but in which these processes are tightly regulated. Recently we could show that HMGI(Y) is expressed in the normal human placenta, where it is localized in the nuclei of villous cytotrophoblast, in the anchoring villi at the implantation site and in extravillous (intermediate) trophoblast invading the maternal decidua. In contrast, the majority of the nuclei of the villous syncytiotrophoblast, a terminally differentiated tissue, was negative. The purpose of this study was to investigate the expression pattern of HMGI(Y) in gestational trophoblastic diseases (GTD), which has not been studied so far. To analyze the expression of HMGI(Y), we performed immunohistochemistry on a total of 29 cases of GTD, including 21 hydatidiform moles and 8 choriocarcinomas. Hydatidiform moles showed a positivity for HMGI(Y) in villous cytotrophoblast and in areas of the trophoblast proliferations on the villous surface; villous syncytiotrophoblast was negative. The choriocarcinomas showed strong immunoreactivity in all cases. The expression pattern of HMGI(Y) in gestational trophoblastic diseases indicates that it might play a role in the pathogenesis of GTD and might be potentially useful as an additional diagnostic marker for such lesions.

TonEBP is inhibited by RNA helicase A via interaction involving the E′F loop

TonEBP [TonE (tonicity-responsive enhancer)-binding protein] is a transcriptional activator of the Rel family like NF-kappaB (nuclear factor kappaB) and NFAT (nuclear factor of activated T-cells). TonEBP plays a key role in the protection of cells in the kidney medulla from the deleterious effects of hyperosmolality. This is achieved by enhancing expression of HSP70 (heat-shock protein 70) and other genes whose products drive cellular accumulation of organic osmolytes. TonEBP is stimulated by ambient hypertonicity via multiple pathways that regulate nuclear translocation and transactivation. In the present paper, we report that TonEBP is associated in vivo with RHA (RNA helicase A). The N- and C-termini of RHA bound the E'F loop of the DNA-binding domain of TonEBP. The interaction was not affected by DNA binding or dimerization of TonEBP. Overexpression of RHA inhibited the activity of TonEBP; however, catalytic activity of RHA was dispensable for the inhibition. When the ambient tonicity was raised, the TonEBP-RHA interaction decreased, suggesting that dissociation of RHA is a pathway to stimulate TonEBP. We conclude that the E'F loop of TonEBP interacts with RHA like NFAT and NF-kappaB interact with AP1 (activator protein 1) and the high-mobility group protein HMG-I(Y) respectively. While RHA interacts with and stimulates other transcription factors such as CREB (cAMP-response-element-binding protein), NF-kappaB and mineralocorticoid receptor, it inhibits TonEBP.

Pleomorphic adenoma (benign mixed tumor) of the breast: A case report and review of the literature

We report a case of pleomorphic adenoma (benign mixed tumor) of the breast, which is an extremely rare location for this tumor. Examination of a 55-year-old woman unexpectedly revealed a mass measuring 0.8 cm in diameter in the subareolar region of the right breast. Excisional biopsy was performed, and the tumor histologically showed pleomorphic adenoma composed of duct epithelial cells, myoepithelial cells, and a myxochondroid matrix. Immunohistochemically, duct epithelial cells were positive for the estrogen receptor, but negative for the progesterone receptor. The nuclei of the spindle and myoepithelial cells were immunoreactive for HMGI-C and HMGI(Y) proteins, indicating a histogenesis similar to pleomorphic adenoma of the salivary glands. Interphase fluorescence in situ hybridization performed on paraffin-embedded tissue sections with 12q15 probes and a 6p21 probe demonstrated no chromosomal rearrangement. Sixty-nine cases of this type of tumor arising in the breast have been described previously. Using imaging procedures, the tumor has occasionally been misdiagnosed as malignant clinically and even pathologically in frozen section diagnosis. Careful diagnosis based on paraffin sections is required to avoid unnecessary aggressive surgery, and pathologists should include pleomorphic adenoma in the differential diagnosis of a demarcated, juxtaareolar, small hard mass.

Modulation of DNA Conformations Through the Formation of Alternative High-order HU–DNA Complexes

HU is an abundant, highly conserved protein associated with the bacterial chromosome. It belongs to a small class of proteins that includes the eukaryotic proteins TBP, SRY, HMG-I and LEF-I, which bind to DNA non-specifically at the minor groove. HU plays important roles as an accessory architectural factor in a variety of bacterial cellular processes such as DNA compaction, replication, transposition, recombination and gene regulation. In an attempt to unravel the role this protein plays in shaping nucleoid structure, we have carried out fluorescence resonance energy transfer measurements of HU–DNA oligonucleotide complexes, both at the ensemble and single-pair levels. Our results provide direct experimental evidence for concerted DNA bending by HU, and the abrogation of this effect at HU to DNA ratios above about one HU dimer per 10–12 bp. These findings support a model in which a number of HU molecules form an ordered helical scaffold with DNA lying in the periphery. The abrogation of these nucleosome-like structures for high HU to DNA ratios suggests a unique role for HU in the dynamic modulation of bacterial nucleoid structure.

The HMG-I oncogene causes highly penetrant, aggressive lymphoid malignancy in transgenic mice and is overexpressed in human leukemia.

HMG-I/Y is overexpressed in human cancer, although a direct role for this gene in transformation has not been established. We generated transgenic mice with HMG-I targeted to lymphoid cells. All seven informative founder HMG-I mice developed aggressive lymphoma by a mean age of 4.8 months. Tumors express T-cell markers and are transplantable. We also demonstrate that HMG-I mRNA and protein are increased in human acute lymphocytic leukemia samples. Our results show that HMG-I functions as an oncogene and suggest that it contributes to the pathogenesis of leukemia and other cancers with increased HMG-I expression.

High mobility group protein HMGI(Y) enhances tumor cell growth, invasion, and matrix metalloproteinase-2 expression in prostate cancer cells.

The high mobility group protein HMGI(Y) has oncogenic properties and correlates with an aggressive phenotype in prostate cancer. The molecular mechanisms involved in transformation associated with HMGI(Y) overexpression remain unknown. The HMG-I isoform was transfected and overexpressed in nonmetastatic Dunning prostate cancer cells (G cells) without detectable HMGI(Y). The assays of cell proliferation, tumor formation, in vitro invasion, and cDNA microarray were performed to assess the effect of HMGI(Y) overexpression in the transfected G cells. Overexpression of HMG-I in G cells significantly increases cell proliferation and tumor growth and also modestly enhances in vitro invasion compared to mock transfectant. cDNA microarray revealed that expression of the matrix metalloproteinase-2 (MMP-2) proform was increased eightfold in G cells overexpressing HMG-I. Overexpression of HMG-I in prostate cancer cells enhances cell growth, invasion, and expression of the proform of MMP-2, which may initiate early steps involved in the metastatic cascade.

Expression of the high mobility group proteins HMGI(Y) correlates with malignant progression in Barrett's metaplasia.

Expression of the high mobility group proteins HMGI(Y) has been shown to be a marker of malignancy in thyroid and pancreatic lesions and to correlate significantly with malignant progression in the colon. The aim of this study was to determine whether HMGI(Y) expression is associated with malignant progression in Barrett's metaplasia (BM). Immunoperoxidase staining for HMGI(Y) was performed on sections of formalin-fixed paraffin-embedded endoscopic esophageal biopsies from 42 patients with BM. These consisted of 19 biopsies negative for dysplasia (ND), 16 with low-grade dysplasia (LGD)/indeterminate for dysplasia (IND), and 7 with high-grade dysplasia (HGD)/adenocarcinoma (CA). The percentage of positive cells was recorded, and nuclear HMGI(Y) immunoreactivity in >10% of the cells was considered positive. Statistical analysis was performed using Fisher's exact test. Positive HMGI(Y) staining was detected in 2 of 19 (11%) cases ND, 5 of 16 (30%) LGD/IND cases, and 7 of 7 (100%) HGD/CA cases. Biopsies with HGD/CA were significantly more likely to be positive for HMGI(Y) than biopsies ND (P < 0.0001) or with LGD/IND (P = 0.0046). We conclude that HMGI(Y) expression is significantly associated with malignant progression in BM. Additional studies are needed to determine whether BM biopsies that are ND or LGD/IND and positive for HMGI(Y) are more likely to progress to adenocarcinoma.

AT-hook proteins stimulate induction of senescence markers triggered by 5-bromodeoxyuridine in mammalian cells

5-Bromodeoxyuridine (BrdU) induces a phenomenon similar to cellular senescence in mammalian cells. To address an underlying molecular mechanism in this phenomenon, we assessed the role of AT-hook proteins that bind to the minor grooves of specific AT-rich sequences. We expressed DsRed-tagged HMGI, MATH2, and MATH20 proteins in HeLa cells in a doxycycline dependent manner. Modest expression of these proteins revealed no apparent effect on the cells although high levels of expression were toxic to the cells. In contrast, their modest expression in the presence of low concentrations of BrdU similarly and dose-dependently induced senescence markers examined, although the same concentrations of BrdU alone showed no obvious effect. In both cases, DsRed fluorescence was mainly observed as foci or intense dots on Hoechst 33342-staining regions. These distribution patterns were not changed by addition of BrdU. Since AT-hook domains can displace chromatin compacting proteins pre-bound on AT-rich sequences, these results suggest that chromatin unpacking is one of the factors stimulating expression of the senescence markers in human cells.

Expression of the high-mobility group protein HMGI(Y) in human trophoblast: potential role in trophoblast invasion of maternal tissue.

The high-mobility group protein HMGI(Y) is a member of a family of non-histone chromosomal proteins, which have been implicated in the regulation of inducible gene transcription, integration of retroviruses into chromosomes and induction of neoplastic transformation and metastatic progression in cancer cells. The human trophoblast is a tissue that shares proliferation capacity and invasiveness with neoplastic tissues, but in which these processes are tightly regulated. In the present study, we analyzed the expression of HMGI(Y) in the human placenta using immunohistochemistry. We found expression of HMGI(Y), with nuclear localization, in the villous cytotrophoblast (vCT), which is a highly proliferative cell type. In contrast, the majority of the nuclei of the villous syncytiotrophoblast, a terminally differentiated tissue, was negative. Interestingly, expression of HMGI(Y) was strongest in anchoring villi at the implantation site and in extravillous (intermediate) trophoblast (EVT) invading the maternal decidua. As vCT cells differentiate to become EVT, the HMGI(Y) protein appears to switch from a nuclear to a cytoplasmic localization. Expression of HMGI(Y) in isolated trophoblast populations in primary cell culture was also confirmed using Western-blot analysis. This study shows for the first time expression and localization of HMGI(Y) in the subpopulations of placental tissue.

Hmga1 is required for normal sperm development.

The Hmgi protein family of chromosomal architectural factors is extensively studied for its roles in embryogenesis and its association with benign mesenchymal tumors. Although the biochemical function of Hmga1 has been studied in vitro, to provide in vivo insight into its biological function, a targeted disruption of Hmga1 was initiated. Chimeric founder mice were derived from embryonic stem (ES) cells harboring a targeted mutation in a single Hmga1 allele. These 14 different chimeric founders produced 494 black progeny. Since none of these 494 progeny were agouti, none of them were derived from ES cells. Control injections of the wild-type ES cell lines resulted in ES cell derived agouti mice, indicating that the ES cells were totipotent. Therefore, our results indicate that one intact Hmga1 allele was not sufficient for germ-line transmission of the ES cells. Seven chimeric founder mice that were examined histologically demonstrated aberrant regions in their reproductive organs. Aberrant regions of seminiferous tubules were reduced in diameter, demonstrated vacuolated Sertoli cells, and had an absolute deficiency of sperm. While the Hmga1(+/-) ES cells were shown to contribute to the formation of the epididymides, they did not significantly contribute to the testes of chimeric founder mice. No sperm isolated from any of the Hmga1(+/-) chimeric mice were shown to arise from the ES cells, as none of them contained the targeted disruption of the Hmga1 gene. Our results suggest that both alleles of Hmga1 are required for normal sperm production in the mouse.

HMG-I(Y) and the CBP/p300 coactivator are essential for human papillomavirus type 18 enhanceosome transcriptional activity.

A strong epithelial specific enhancer drives transcription of the human papillomavirus type 18 (HPV18) oncogenes. Its activity depends on the formation of a higher-order nucleoprotein complex (enhanceosome) involving the sequence-specific JunB/Fra2 transcription factor and the HMG-I(Y) architectural protein. Here we show that proteins from HeLa cell nuclear extract cover almost all of the HPV18 enhancer sequences and that it contains seven binding sites for the purified HMG-I(Y) protein, providing evidence for a tight nucleoprotein structure. Binding of HMG-I(Y) and the AP1 heterodimer from HeLa nuclear extract to overlapping sites of the core enhanceosome is cooperative. The integrity of this specific HMG-I(Y) binding site is as essential as the AP1 binding site for the enhancer function, indicating the fundamental role played by this architectural protein. We demonstrate that the CBP/p300 coactivator is recruited by the HPV18 enhanceosome and that it is limiting for transcriptional activation, since it is sequestered by the adenovirus E1A protein and by the JunB/Fra2 positive factor in excess. We show the involvement of JunB and p300 in vivo in the HPV18 transcription by chromatin immunoprecipitation of HPV18 sequences in HeLa cells.

Expression of the high mobility group proteins HMGI(Y) correlates with malignant progression in Barrett metaplasia

Expression of the high mobility group proteins HMGI(Y) correlates with malignant progression in Barrett metaplasia

Nuclear matrix localization of high mobility group protein I(Y) in a transgenic mouse model for prostate cancer.

Nuclear shape and the underlying nuclear structure, the nuclear matrix in cancer cells. Since the NM composition is considered to maintain nuclear shape and architecture, nuclear matrix proteins (NMPs) may be involved in transformation. Our laboratory has recently characterized a subset of NMPs that are associated with prostate cancer development in the transgenic adenocarcinoma of mouse prostate (TRAMP) model. One of the identified NMPs, E3E, has a similar molecular weight (22 kDa) with a protein known as HMGI(Y). HMGI(Y) belongs to a group of non-histone and chromatin-associated proteins, high-mobility-group (HMG) proteins, and it has been shown to associate with the NM. HMGI(Y) has been reported to be elevated in different types of cancer including prostate cancer. In this study, we examined the expression of HMGI(Y) protein in the NMP composition of the TRAMP model during the progression from normal to neoplasia. The expression of HMGI(Y) in the NMP extracts of three prostatic epithelial cell lines derived from a 32-week TRAMP mouse: TRAMP-C1, TRAMP-C2, and TRAMP-C3 was also examined. Using both one-dimensional and high-resolution two-dimensional immunoblot analyses, we found that: (i) HMGI(Y) is a nuclear matrix protein expressed as two protein bands with MW of 22-24 kDa and (ii) HMGI(Y) expression is correlated with neoplastic and malignant properties in late stage TRAMP prostate tumors. Overall, these findings support the evidence that HMGI(Y) can be utilized as a marker and prognostic tool for prostate cancer.

Diagnostic significance of high mobility group I(Y) protein expression in intraductal papillary mucinous tumors of the pancreas.

Overexpression of the high mobility group I(Y), [HMGI(Y)], gene/proteins has been demonstrated in many types of human malignancies, suggesting that HMGI(Y) may play a vital role in the oncogenic transformation of cells. To analyze HMGI(Y) expression in intraductal papillary mucinous tumor (IPMT) of the pancreas to verify whether determination of the HMGI(Y) expression level could provide any diagnostic advantages in the pathological diagnosis of IPMT. Thirty-three different lesions from 25 patients with IPMT, including 20 with mild dysplasia, 7 with moderate dysplasia, and 6 with carcinoma, were analyzed immunohistochemically with use of an HMGI(Y)-specific antibody. Immunohistochemical analysis revealed that, although no significant immunoreactivity was found in cases of normal pancreatic duct or mild dysplasia, 28.6% (2/7) of moderate dysplasia showed multifocal immunoreactivity with moderate intensity. In contrast, in 50% (3/6) of the cases of carcinoma, intense multifocal or diffuse immunoreactivity occurred, almost equivalent to that observed in cases of duct cell carcinoma, whereas in the remaining 3 cases of carcinoma only a faint focal immunoreactivity occurred. Histologic examination revealed that these HMGI(Y)-positive carcinomas had an invasive growth pattern, whereas the HMGI(Y)-negative carcinomas were either carcinomas in situ or tumors with minimal invasion. Thus, an increased expression level of HMGI(Y) proteins was closely associated with the malignant phenotype in IPMT. On the basis of these findings, we propose that HMGI(Y) proteins could play an important role(s) in a multistage process of carcinogenesis of IPMT and that the HMGI(Y) protein level could serve as a potential diagnostic marker, which may enable the identification of tumor cells with potential to be biologically malignant.

Changes in the Mechanism of DNA Integration in Vitro Induced by Base Substitutions in the HIV-1 U5 and U3 Terminal Sequences

We have reconstituted concerted human immunodeficiency virus type 1 (HIV-1) integration with specially designed mini-donor DNA, a supercoiled plasmid acceptor, purified bacterial-derived HIV-1 integrase (IN), and host HMG-I(Y) protein (Hindmarsh, P., Ridky, T., Reeves, R., Andrake, M., Skalka, A. M., and Leis, J. (1999) J. Virol. 73, 2994-3003). Integration in this system is dependent upon the mini donor DNA having IN recognition sequences at both ends and the reaction products have all of the features associated with integration of viral DNA in vivo. Using this system, we explored the relationship between the HIV-1 U3 and U5 IN recognition sequences by analyzing substrates that contain either two U3 or two U5 terminal sequences. Both substrates caused severe defects to integration but with different effects on the mechanism indicating that the U3 and the U5 sequences are both required for concerted DNA integration. We have also used the reconstituted system to compare the mechanism of integration catalyzed by HIV-1 to that of avian sarcoma virus by analyzing the effect of defined mutations introduced into U3 or U5 ends of the respective wild type DNA substrates. Despite sequence differences between avian sarcoma virus and HIV-1 IN and their recognition sequences, the consequences of analogous base pair substitutions at the same relative positions of the respective IN recognition sequences were very similar. This highlights the common mechanism of integration shared by these two different viruses.

High mobility group I (Y) proteins bind HIPK2, a serine-threonine kinase protein which inhibits cell growth.

The HMGI proteins (HMGI, HMGY and HMGI-C) have an important role in the chromatin organization and interact with different transcriptional factors. The HMGI genes are expressed at very low levels in normal adult tissues, whereas they are very abundant during embryonic development and in several experimental and human tumours. In order to isolate proteins interacting with the HMGI(Y) proteins, a yeast two-hybrid screening was performed using the HMGI(Y) protein as bait. This analysis led to the isolation of homeodomain-interacting protein kinase-2 (HIPK2), a serine/threonine nuclear kinase. HIPK2 co-immunoprecipitates with the HMGI(Y) protein in 293T cells. The interaction between HIPK2 and HMGI(Y) occurs through the PEST domain of HIPK2 and it is direct because in vitro translated HIPK2 binds HMGI(Y). We also show that HIPK2 is able to phosphorylate the HMGI(Y) protein by an in vitro kinase assay. In order to understand a possible role of HIPK2 gene in cell growth we performed a colony assay which showed an impressive HIPK2 inhibitory effect on normal thyroid cells. Flow cytometric analysis would indicate the block of cell growth at the G2/M phase of the cell cycle. Since normal thyroid cells do not express detectable HMGI(Y) protein levels, we assume that the HIPK2 inhibitory effect is independent from the interaction with the HMGI(Y) protein.