HMGA1 Protein Research Articles

Comment Re: HMGA2 Is a Negative Regulator of DNA-PK Pathway

To the Editor: Recently, Li and colleagues ([1][1]) have presented evidence for the impairment of nonhomologous and joining repair (NHEJ) of DNA damage by the chromatin binding protein HMGA2. In this interesting article, the cytogenetic stability of fibroblasts transfected by a construct encoding

Clinical pathological significance of HMGA1 and HMGA2 protein expression in esophageal squamous cell carcinoma

Clinical pathological significance of HMGA1 and HMGA2 protein expression in esophageal squamous cell carcinoma

Effects of small interfering RNA specific to stably transfected HMGA1 gene on biological characters of ovarian carcinoma cells

To investigate the effects of biological characters of the human ovarian cell line (OVCAR) by stable transfection short hairpin RNA into the target HMGA1 gene. Experiments were divided into two groups: transfected the OVCAR cells with pSilence4.1-CMV-Hs plasmid as group A, while transfected OVCAR cells with pSilence4.1-CMV-Hn plasmid as group B, in which stably transfected cells were gained by antibiotic screening. The comparative expressions of HMGA1 were detected by RT-PCR and western blot. Methyl thiazolyl tetrazolium (MTT) method was applied to measure cell proliferation and at the same time, the cell growth curve was also be mapped. Vitro invasion assay was used to observe the invasion ability of the cancer cells, and the tumor growth of the nude mice inoculated of tumor cells were compared with before and after transfection. In group A, the expression level of mRNA and protein HMGA1 gene in OVCAR cells were remarkably reduced before and after the stable transfected with HMGA1 siRNA, in which the percents of mRNA expression were [(86.3 ± 2.7)% vs. (35.8 ± 3.1)%, P < 0.05], the expression of protein were [(68.6 ± 2.8)% vs. (22.3 ± 4.2)%, P < 0.05)]. The OVCAR cell growth in stable transfection status was more significantly decreased than that in non-transfection status (P < 0.05). In group B, there were no statistical difference in the expression of HMGA1 siRNA, protein and the cell growth between before and after transfection states (P > 0.05). The invasion cell numbers were reduced from before to after transfection state in group A [(53 ± 6) vs. (21 ± 6), P < 0.05], while there was no significant difference in group B [(51 ± 6) vs. (47 ± 8), P > 0.05]. After inoculated transfected cells into nude mice, it took (6.0 ± 0.9) days to grow the planed tumors in group A, which was much shorter than that (12.3 ± 3.9) days in group B (P < 0.05). After 5 weeks, the tumor weight and volume in group A was were significantly lower than those in group B [(0.8 ± 0.3) g vs. (2.1 ± 0.4) g, (205 ± 34) mm(3) vs. (987 ± 82) mm(3), all P < 0.05]. HMGA1 siRNA could remarkably reduce the expression of HMGA1 gene in ovarian cell and also inhabit the ovarian cell growth.

HMGA1 protein expression in familial breast carcinoma patients

HMGA protein overexpression is associated with a highly malignant phenotype and it is also causally related to neoplastic cell transformation. Our previous results have shown that HMGA1 was not expressed in normal breast tissue whereas HMGA1 staining was intense in 25% of hyperplastic lesions with cellular atypia and in 60% of sporadic ductal carcinomas. Moreover, HMGA1 protein levels were significantly correlated with c-Erb-B2 expression. These results suggested HMGA1 expression as a novel prognostic factor in breast ductal carcinomas.In order to investigate whether the HMGA1 detection might have a prognostic role also for inherited breast carcinomas we have analysed the expression of the HMGA1 proteins in 116 breast familial carcinomas associated with BRCA1 or BRCA2 or negative for mutations in both genes (BRCAX). HMGA1 expression was weakly positive in 23 (20%), moderately positive in 34 (29%) and strongly positive in 20 (17%) breast carcinomas, and was not detected in 39 of them (34%). Statistical analysis of the immunostaining data showed that HMGA1 was significantly overexpressed, with a more intense staining, in BRCA2 (p=0.0009) and BRCAX (p=0.0134) patients compared to BRCA1 ones.Furthermore, in BRCA2 positive patients, the expression of HMGA1 seems to correlate with a favourable prognosis with a median overall survival of 65months and a 5-year survival rate of 80% for HMGA1-negative patients, while median overall survival in the HMGA1-positive subsets was not reached with 5-year survival rates ranging from 84% to 100% of patients (p=0.0198). Conversely, no correlation was found between HMGA1 expression and overall survival in patients carrying inherited mutations in the BRCA1 and in BRCAX patients.

HMGA1 Levels Influence Mitochondrial Function and Mitochondrial DNA Repair Efficiency

HMGA chromatin proteins, a family of gene regulatory factors found at only low concentrations in normal cells, are almost universally overexpressed in cancer cells. HMGA proteins are located in the nuclei of normal cells except during the late S/G(2) phases of the cell cycle, when HMGA1, one of the members of the family, reversibly migrates to the mitochondria, where it binds to mitochondrial DNA (mtDNA). In many cancer cells, this controlled shuttling is lost and HMGA1 is found in mitochondria throughout the cell cycle. To investigate the effects of HMGA1 on mitochondria, we employed a genetically engineered line of human MCF-7 cells in which the levels of transgenic HMGA1 protein could be reversibly controlled. "Turn-ON" and "turn-OFF" time course experiments were performed with these cells to either increase or decrease intracellular HMGA1 levels, and various mitochondrial changes were monitored. Results demonstrated that changes in both mtDNA levels and mitochondrial mass inversely paralleled changes in HMGA1 concentrations, strongly implicating HMGA1 in the regulation of these parameters. Additionally, the level of cellular reactive oxygen species (ROS) increased and the efficiency of repair of oxidatively damaged mtDNA decreased as consequences of elevated HMGA1 expression. Increased ROS levels and reduced repair efficiency in HMGA1-overexpressing cells likely contribute to the increased occurrence of mutations in mtDNA frequently observed in cancer cells.

The maize HMGA protein is localized to the nucleolus and can be acetylated in vitro at its globular domain, and phosphorylation by CDK reduces its binding activity to AT-rich DNA

The high mobility group (HMG) proteins are nonhistone chromosomal proteins that participate in diverse nuclear activities including chromatin structure and gene regulation. We previously studied the biochemistry of the maize HMGA protein and its role in transcriptional regulation during maize endosperm development. Here, we extended our study and showed that a strong binding of ZmHMGA to AT-rich DNA requires at least three AT-hook motifs; two motifs showed a significant reduction whereas a single motif was not sufficient for binding. CDK phosphorylation sites situated between AT-hook3 and AT-hook4 were strongly phosphorylated by a SUC1-associated kinase; no in vitro phosphorylation is evident for the AtHMGA protein. Phosphorylation of ZmHMGA reduced its binding to AT-rich DNA in vitro. The maize HMGA protein fused to GFP was localized in the nucleus of transgenic Arabidopsis plants tending to concentrate within the nucleolus. Localization to the nucleolus was conferred by the C-terminal portion of the protein containing the AT-hooks. ZmHMGA was acetylated in vitro on its N-terminal globular domain by the human PCAF acetyltransferase. Our results suggest that ZmHMGA participates in nucleolar function and that its role may be regulated posttranslationally by phosphorylation and acetylation.

Interaction between HMGA1 and Retinoblastoma Protein Is Required for Adipocyte Differentiation

It is generally accepted that the regulation of adipogenesis prevents obesity. However, the mechanisms controlling adipogenesis have not been completely defined. We have previously demonstrated that HMGA1 proteins play a critical role in adipogenesis. In fact, suppression of HMGA1 protein synthesis by antisense technology dramatically increased growth rate and impaired adipocyte differentiation in 3T3-L1 cells. Furthermore, we showed that HMGA1 strongly potentiates the capacity of the CCAAT/enhancer-binding protein beta (C/EBPbeta) transcriptional factor to transactivate the leptin promoter, an adipocytic-specific promoter. In this study we demonstrate that HMGA1 physically interacts with retinoblastoma protein (RB), which is also required in adipocyte differentiation. Moreover, we show that RB, C/EBPbeta, and HMGA1 proteins all cooperate in controlling both Id1 and leptin gene transcriptions, which are down- and up-regulated during adipocyte differentiation, respectively. We also demonstrate that HMGA1/RB interaction regulates CDC25A and CDC6 promoter activities, which are induced by E2F-1 protein during early adipocyte differentiation, by displacing HDAC1 from the RB-E2F1 complex. Furthermore, by using Hmga1(-/-) embryonic stem cells, which failed to undergo adipocyte differentiation, we show the crucial role of HMGA1 proteins in adipocyte differentiation due to its pivotal involvement in the formation of the RB-C/EBPbeta complex. Altogether these data demonstrate a key role of the interaction between HMGA1 and RB in adipocyte differentiation.

The High Mobility Group Protein HMGA2: A Co-Regulator of Chromatin Structure and Pluripotency in Stem Cells?

The small, chromatin-associated HMGA proteins contain three separate DNA binding domains, so-called AT hooks, which bind preferentially to short AT-rich sequences. These proteins are abundant in pluripotent embryonic stem (ES) cells and most malignant human tumors, but are not detectable in normal somatic cells. They act both as activator and repressor of gene expression, and most likely facilitate DNA architectural changes during formation of specialized nucleoprotein structures at selected promoter regions. For example, HMGA2 is involved in transcriptional activation of certain cell proliferation genes, which likely contributes to its well-established oncogenic potential during tumor formation. However, surprisingly little is known about how HMGA proteins bind DNA packaged in chromatin and how this affects the chromatin structure at a larger scale. Experimental evidence suggests that HMGA2 competes with binding of histone H1 in the chromatin fiber. This could substantially alter chromatin domain structures in ES cells and contribute to the activation of certain transcription networks. HMGA2 also seems capable of recruiting enzymes directly involved in histone modifications to trigger gene expression. Furthermore, it was shown that multiple HMGA2 molecules bind stably to a single nucleosome core particle whose structure is known. How these features of HMGA2 impinge on chromatin organization inside a living cell is unknown. In this commentary, we propose that HMGA2, through the action of three independent DNA binding domains, substantially contributes to the plasticity of ES cell chromatin and is involved in the maintenance of a un-differentiated cell state.

Single-Chromophore-Based Photoswitchable Nanoparticles Enable Dual-Alternating-Color Fluorescence for Unambiguous Live Cell Imaging

We have developed a class of spiropyran dyes and their fluorescence colors can be reversibly photoswitched from red to green, blue, or nearly dark, thus alternating between two colors. Such individual dyes emit either one color or the other but not both simultaneously. Nanoparticles enabled with these photoswitchable dyes, however, emit either one pure color or a combination of both colors because the nanoparticle fluorescence originates from multiple dyes therein. As a result, the nanoparticle shines >30 times brighter than state-of-the-art organic dyes such as fluorescein. Interestingly, these copolymer nanoparticles exhibit tunable nonspecific interactions with live cells, and nanoparticles containing properly balanced butyl acrylate and acrylamide monomers render essentially very little nonspecific binding to live cells. Decorated with HMGA1 protein, these optically switchable dual-color nanoparticles undergo endocytosis and unambiguously identify themselves from fluorescence interference including autofluorescence, thus enabling a new tool for live cell imaging.

Regulation of microRNA expression by HMGA1 proteins

The High Mobility Group proteins HMGA1 are nuclear architectural factors that play a critical role in a wide range of biological processes. Since recent studies have identified the microRNAs (miRNAs) as important regulators of gene expression, modulating critical cellular functions such as proliferation, apoptosis and differentiation, the aim of our work was to identify the miRNAs that are physiologically regulated by HMGA1 proteins. To this purpose, we have analysed the miRNA expression profile of mouse embryonic fibroblasts (MEFs) carrying two, one or no Hmga1 functional alleles using a microarray (miRNA microarray). By this approach, we found a miRNA expression profile that differentiates Hmga1-null MEFs from the wild-type ones. In particular, a significant decrease in miR-196a-2, miR-101b, miR-331 and miR-29a was detected in homozygous Hmga1-knockout MEFs in comparison with wild-type cells. Consistently, these miRNAs are downregulated in most of the analysed tissues of Hmga1-null mice in comparison with the wild-type mice. ChIP assay shows that HMGA1 is able to bind regions upstream of these miRNAs. Moreover, we identified the HMGA2 gene product as a putative target of miR-196a-2, suggesting that HMGA1 proteins are able to downregulate the expression of the other member of the HMGA family through the regulation of the miR-196a-2 expression. Finally, ATXN1 and STC1 gene products have been identified as targets of miR-101b. Therefore, it is reasonable to hypothesize that HMGA1 proteins are involved in several functions by regulating miRNA expression.

HMGA2, MicroRNAs, and Stem Cell Aging

Mammalian aging results from a replicative decline in the function of somatic stem cells and other self-renewing cells. Recent studies (Monzen et al., 2008; Nishino et al., 2008; Sanna et al., 2008; Weedon et al., 2008) link a chromatin-associated protein, HMGA2, to development, height, and mouse stem cell aging during late fetal development and young adulthood.

A microRNA encoded in a highly conserved part of the mammalian HMGA2 gene

The high mobility group protein HMGA2 plays an important role as a chromatin component of stem cells and as a protein causally related to the development of a variety of benign tumors (e.g., uterine leiomyomas, lipomas, and pleomorphic adenomas of the salivary glands). Herein, the existence of a highly conserved region within intron 3 of HMGA2 encoding a microRNA is described. The co-expression with HMGA2 suggests that as an intronic microRNA, this microRNA may cooperate with HMGA2 in its physiological and/or aberrant functions.

Abstract 3608: High Mobility Group A1 Protein - A New Regulator for Peroxisome Proliferator-Activated Receptor Gamma Transcriptional Repression in Human Aortic Smooth Muscle Cells

The study aimed to identify new nuclear cofactors for PPARgamma (peroxisome proliferator-activated receptor gamma)-dependent gene transcription in human aortic smooth muscle cells (HASMC) in order to develop new PPARgamma-ligands with improved clinical safety in the absence of deleterious cardiovascular side effects. Using an Oligo GEArray® Human Nuclear Receptors and Coregulators Microarray for gene expression profiling, we identified the transcriptional regulator and chromatin modifying High Mobility Group (HMG) A1 protein highly expressed in unstimulated HASMC. PPARgamma-dependent gene regulation was studied by analysis of PMA-induced MMP-9 (matrix metalloproteinase 9) expression ± pioglitazone (pio 10μM). PMA (50ng/ml) stimulated MMP-9 mRNA expression by 46.3±22.3-fold (p&lt;0.05 vs. vehicle) which was markedly blocked by pio (10μM: 17.4±4.8-fold vs. PMA alone p&lt;0.05). Pio also blocked PMA-induced MMP-9 promoter activity by 45% in transactivation assays in HEK293 using a pGL3-MMP-9 2.2 kb construct. To evaluate the role of HMGA1, gene silencing experiments with siRNA for HMGA1 were performed (91 % in HASMC and 80.2% in HEK293 reduction of HMGA1 protein expression). HMGA1 siRNA completely abolished PPARgamma-mediated MMP9-mRNA repression (control siRNA: pio-mediated MMP-9 regulation vs. PMA alone: −66.8 % in HASMC and −59.3% in HEK293 p&lt;0.01; HMGA1 siRNA: pio-mediated MMP-9 regulation vs. PMA alone: +10.7 % in HASMC and +14.7% in HEK293 vs. PMA alone; p=n.s.). Knockdown of HMGA1 expression reverse trans-repression of MMP9 by PPARgamma in HASMCs. By using ChIP assay we could demonstrate that pio-induced PPARgamma activation leads to a potent recruitment of PPARgamma (3.0 fold vs.1.15 fold PMA alone) and HMGA1 complexes (1.24 fold vs. 0.0 fold PMA alone) to the MMP9 promoter in HASMC. In consonance with reduced promoter activity, RNA-Polymerase II was removed from the MMP9 promoter by pio (0.08 fold vs 1.04 fold PMA alone). In conclusion, HMGA1 is required for PPARgamma-mediated repression of MMP-9 gene transcription. Ligand-induced HMGA1-PPARgamma interactions might be an important determinant for ligand-specific anti-atherosclerotic actions.

HMGA1 protein is a novel target of the ATM kinase

The high mobility group HMGA1 protein belongs to a family of architectural factors that play a role in chromosomal organisation and gene transcription regulation. HMGA1 overexpression represents a common feature of human malignant tumours and is causally associated with neoplastic transformation and metastatic progression. Recently, HMGA1 expression has been correlated with the presence of chromosomal rearrangements and suggested to promote genomic instability. Here, we report a novel interaction between HMGA1 protein and the ataxia-telangiectasia mutated (ATM) kinase, the major key player in the cellular response to DNA damage caused by several agents such as ionising radiation (IR). We identified an SQ motif on HMGA1, which is effectively phosphorylated by ATM in vitro and in vivo. Interestingly, confocal microscopy revealed that HMGA1 colocalises with the activated form of ATM (ATM S1981p). Moreover, HMGA1 ectopic expression decreases cell survival following exposure to IR as assessed by clonogenic survival in MCF-7 cells, further supporting the hypothesis that HMGA1 might act as a downstream target of the ATM pathway in response to DNA damage.

A crucial role of a high mobility group protein HMGA2 in cardiogenesis

The high mobility group (HMG) of nuclear proteins regulates expression of many genes through architectural remodelling of the chromatin structure, and formation of multiprotein complexes on promoter/enhancer regions. This leads to the active transcription of their target genes. Here we show that HMGA2, a member of the HMGA sub-family of HMG proteins, has a critical function in cardiogenesis. Overexpression of HMGA2 enhanced, whereas siRNA-mediated knockdown of HMGA2 blocked, cardiomyocyte differentiation of the embryonal carcinoma cell line P19CL6. Moreover, overexpression of a dominant-negative HMGA2 or morpholino-mediated knockdown of HMGA2 expression blocked normal heart formation in Xenopus laevis embryos, suggesting that HMGA2 has an important role in cardiogenesis both in vitro and in vivo. Mechanistically, HMGA2 associated with Smad1/4 and showed synergistic trans-activation of the gene for a cardiac transcription factor Nkx2.5; a conserved HMGA2 binding site was required for the promoter activity of Nkx2.5 gene, both in P19CL6 cells and in transgenic Xenopus embryos. Thus, HMGA2 is a positive regulator of Nkx2.5 gene expression and is essential for normal cardiac development.

The HMGA proteins: A myriad of functions (Review)

The 'high mobility group' HMGA protein family consists of four members: HMGA1a, HMGA1b and HMGA1c, which result from translation of alternative spliced forms of one gene and HMGA2, which is encoded for by another gene. HMGA proteins are characterized by three DNA-binding domains, called AT-hooks, and an acidic carboxy-terminal tail. HMGA proteins are architectural transcription factors that both positively and negatively regulate the transcription of a variety of genes. They do not display direct transcriptional activation capacity, but regulate gene expression by changing the DNA conformation by binding to AT-rich regions in the DNA and/or direct interaction with several transcription factors. In this way, they influence a diverse array of normal biological processes including cell growth, proliferation, differentiation and death. Both HMGA1 and HMGA2 are hardly detectable in normal adult tissue but are abundantly and ubiquitously expressed during embryonic development. In malignant epithelial tumors as well as in leukemia, however, expression of HMGA1 is again strongly elevated to embryonic levels thus leading to ectopic expression of (fetal) target genes. HMGA2 overexpression also has a causal role in inducing neoplasia. Besides overexpression of full length HMGA proteins in different tumors, the HMGA genes are often involved in chromosomal rearrangements. Such translocations are mostly detected in benign tumors of mesenchymal origin and are believed to be one of the most common chromosomal rearrangements in human neoplasia. To provide clarity in the abundance of articles on this topic, this review gives a general overview of the nuclear functions and regulation of the HMGA genes and corresponding proteins.

Detection of high‐mobility group proteins A1 and A2 represents a valid diagnostic marker in post‐pubertal testicular germ cell tumours

The high-mobility group A (HMGA) non-histone chromosomal proteins HMGA1 and HMGA2 are architectural factors. They are abundantly expressed during embryogenesis and in most malignant neoplasias, whereas their expression is low or absent in normal adult tissues. Their over-expression is known to have a causal role in cellular neoplastic transformation. Previous studies from our group have shown that their expression is restricted to specific germinal cells. In this study we have evaluated, by immunohistochemistry, the expression of HMGA1 and HMGA2 in a series of post-pubertal testicular tumours of different histological types, including 30 seminomas, 15 teratomas, 15 embryonal carcinomas and 10 mixed germinal tumours with a prominent yolk sac tumour component. HMGA1 protein expression was detected in all seminomas and embryonal carcinomas analysed, but not in teratomas or yolk sac carcinomas. Conversely, HMGA2 was present only in embryonal carcinomas and yolk sac carcinomas, but not in seminomas or teratomas. The immunohistochemical data were further confirmed by Western blot and, at the mRNA level, by RT-PCR analyses. These findings indicate that HMGA1 and HMGA2 are differently expressed with respect to the state of differentiation of testicular germ cell tumours (TGCTs), with over-expression of both proteins in pluripotential embryonal carcinoma cells and loss of expression of HMGA1 in yolk sac tumours and of both proteins in the mature adult tissue of teratoma areas. Therefore, the different profiles of HMGA1 and HMGA2 protein expression could represent a valuable diagnostic tool in some cases in which the histological differential diagnosis is problematic.

HMGA2 overexpression in non‐small cell lung cancer

Lung cancer is still the leading cause of death from cancer worldwide primarily because of the fact that most lung cancers are diagnosed at advanced stages. Overexpression of the high mobility group protein HMGA2 has been observed in a variety of malignant tumors and often correlates with poor prognosis. Herein, HMGA2 expression levels were analyzed in matching cancerous and non-cancerous lung samples of 17 patients with adenocarcinoma (AC) and 17 patients with squamous cell carcinoma (SCC) with real-time quantitative RT-PCR (qRT-PCR). Transcript levels were compared to results obtained by immunohistochemistry (IHC). HMGA2 expression was detectable by qRT-PCR in all samples tested and varied from 5422 to 16 991 545 copies per 250 ng total RNA in the carcinoma samples and from 289 to 525 947 copies in the non-cancerous tissue samples. In 33/34 non-small cell lung cancer (NSCLC) samples tested, an overexpression of HMGA2 was revealed with statistically highly significant differences between non-neoplastic and tumor samples for both AC (P < 0.0001) as well as for SCC (P < 0.0001). Expression varies strongly and is increased up to 911-fold for AC and up to 2504-fold for SCC, respectively, with statistically significant higher increase in SCC (P < 0.05). The results presented herein indicate that HMGA2 overexpression is a common event in NSCLC and could serve as molecular marker for lung cancer.

Expression of the high mobility group A family member p8 is essential to maintaining tumorigenic potential by promoting cell cycle dysregulation in LβT2 cells

The mechanism by which the HMGA protein p8 facilitates tumorigenesis may be cell cycle dysregulation. Control- (C) LβT2 cells, which express p8, form tumors at a rate five-times faster than p8-knockdown (p8-KD)-LβT2 cells. In association with this heightened tumorigenic potential, p8-expressing C-LβT2 cells avoid G 0/G 1 arrest and become genetically unstable while p8-KD-LβT2 cells arrest in G 0/G 1, become senescent upon overgrowth, and maintain a diploid population. These phenotypic changes correspond to altered cell cycle regulation at the G 1-to-S transition that may be due to p8-mediated changes in expression of the Cip/Kip family members of cell cycle inhibitors, p21, p27, and p57.

From MicroRNA to Carcinogenesis

Misregulation of microRNA (miRNA) function has been implicated in cancer. However, the precise role of miRNAs in tumorigenesis has been unclear. High mobility group A2 protein (Hmga2) is a small, non-histone, chromatin-associated protein found in a number of benign and malignant tumors, where the gene is often truncated at the 3′ end. Mayr et al . now show that it is the loss of the noncoding 3′ untranslated region of the Hmga2 messenger RNA, and specifically of regulator sites for the let-7 miRNA, that causes the overexpression of Hmga2, and that this overexpression contributes to the progression of carcinogenesis both in a tissue culture assay and in nude mice. C. Mayr, M. T. Hemann, D. P. Bartel, Disrupting the pairing between let-7 and Hmga2 enhances oncogenic transformation. Science 315 , 1576-1579 (2007). [Abstract] [Full Text]