The GTPase RAB20 is a HIF target with mitochondrial localization mediating apoptosis in hypoxia.

Hypoxia is a common pathogenic stress, which requires adaptive activation of the Hypoxia-inducible transcription factor (HIF). In concert transcriptional HIF targets enhance oxygen availability and simultaneously reduce oxygen demand, enabling survival in a hypoxic microenvironment. Here, we describe the characterization of a new HIF-1 target gene, Rab20, which is a member of the Rab family of small GTP-binding proteins, regulating intracellular trafficking and vesicle formation. Rab20 is directly regulated by HIF-1, resulting in rapid upregulation of Rab20 mRNA as well as protein under hypoxia. Furthermore, exogenous as well as endogenous Rab20 protein colocalizes with mitochondria. Knockdown studies reveal that Rab20 is involved in hypoxia induced apoptosis. Since mitochondria play a key role in the control of cell death, we suggest that regulating mitochondrial homeostasis in hypoxia is a key function of Rab20. Furthermore, our study implicates that cellular transport pathways play a role in oxygen homeostasis. Hypoxia-induced Rab20 may influence tissue homeostasis and repair during and after hypoxic stress.

Enamel protein regulation and dental and periodontal physiopathology in MSX2 mutant mice.

Signaling pathways that underlie postnatal dental and periodontal physiopathology are less studied than those of early tooth development. Members of the muscle segment homeobox gene (Msx) family encode homeoproteins that show functional redundancy during development and are known to be involved in epithelial-mesenchymal interactions that lead to crown morphogenesis and ameloblast cell differentiation. This study analyzed the MSX2 protein during mouse postnatal growth as well as in the adult. The analysis focused on enamel and periodontal defects and enamel proteins in Msx2-null mutant mice. In the epithelial lifecycle, the levels of MSX2 expression and enamel protein secretion were inversely related. Msx2+/- mice showed increased amelogenin expression, enamel thickness, and rod size. Msx2-/- mice displayed compound phenotypic characteristics of enamel defects, related to both enamel-specific gene mutations (amelogenin and enamelin) in isolated amelogenesis imperfecta, and cell-cell junction elements (laminin 5 and cytokeratin 5) in other syndromes. These effects were also related to ameloblast disappearance, which differed between incisors and molars. In Msx2-/- roots, Malassez cells formed giant islands that overexpressed amelogenin and ameloblastin that grew over months. Aberrant expression of enamel proteins is proposed to underlie the regional osteopetrosis and hyperproduction of cellular cementum. These enamel and periodontal phenotypes of Msx2 mutants constitute the first case report of structural and signaling defects associated with enamel protein overexpression in a postnatal context.

Differential impact of MSX1 and MSX2 homeogenes on mouse maxillofacial skeleton.

Craniofacial development involves a large number of genes involved in a complex time- and site-specific cascade of cellular crosstalk. Msx homeobox genes are expressed very early and have been implicated in multiple signaling processes. However, little is known about their role in postnatal growth and at adult stages. The aim of this study was to compare the patterns of expression of Msx1 and Msx2 during postnatal growth and homeostasis. We used transgenic mice with a knock-in for Msx1 or Msx2. Msx expression was analyzed on whole-mount experiments on heterozygous mice. The results were confirmed by quantitative RT-PCR on mandible and tibia samples. Steady-state levels of Msx2 mRNA were determined at 2 ages, at postnatal day 14 and after 3 months, corresponding to phases of growth and homeostasis, respectively. Consistent with previous findings, the expression profiles of Msx1 and Msx2 overlapped during embryonic development. By contrast, marked differences in the patterns of expression of these 2 genes were observed during the growth phase. Msx1 was found to be expressed in basal bone during postnatal growth. Msx1 was not expressed in alveolar bone, whereas Msx2 was strongly and continually expressed. Msx2 was present in all growth plate cartilages, as previously shown for Msx1. Autopods displayed different patterns of expression during the mouse life cycle, with continuous expression of Msx1 only. Interestingly, both secretory cells (osteoblasts) and cells involved in bone resorption (osteoclasts) were found to be involved in Msx molecular pathways, their precise involvement depending on the anatomical site. The observed patterns correspond to specific sites during growth and constitute landmarks in our understanding of growth-related oral facial dysmorphologies.

Msx and dlx homeogene expression in epithelial odontogenic tumors.

Epithelial odontogenic tumors are rare jaw pathologies that raise clinical diagnosis and prognosis dilemmas notably between ameloblastomas and clear cell odontogenic carcinomas (CCOCs). In line with previous studies, the molecular determinants of tooth development-amelogenin, Msx1, Msx2, Dlx2, Dlx3, Bmp2, and Bmp4-were analyzed by RT-PCR, ISH, and immunolabeling in 12 recurrent ameloblastomas and in one case of CCOC. Although Msx1 expression imitates normal cell differentiation in these tumors, other genes showed a distinct pattern depending on the type of tumor and the tissue involved. In benign ameloblastomas, ISH localized Dlx3 transcripts and inconstantly detected Msx2 transcripts in epithelial cells. In the CCOC, ISH established a lack of both Dlx3 and Msx2 transcripts but allowed identification of the antisense transcript of Msx1, which imitates the same scheme of distribution between mesenchyme and epithelium as in the cup stage of tooth development. Furthermore, while exploring the expression pattern of signal molecules by RT-PCR, Bmp2 was shown to be completely inactivated in the CCOC and irregularly noticeable in ameloblastomas. Bmp4 was always expressed in all the tumors. Based on the established roles of Msx and Dlx transcription factors in dental cell fates, these data suggest that their altered expression is a proposed trail to explain the genesis and/or the progression of odontogenic tumors.

Characterization of human Rab20 overexpressed in exocrine pancreatic carcinoma.

In a large-scale analysis of gene expression in pancreatic cancer, we isolated the homologue of the mouse Rab20. The mouse protein was previously identified during a search for novel Rab proteins, a family of small GTP-binding proteins involved in the regulation of intracellular vesicular transport. The Rab20 protein has no close relationship to any member of the Rab protein subfamily. In contrast to other members, it contains an insertion of 40 amino acids of unknown function and an inversion of 3 amino acids at the position corresponding to codon 61 in p21ras proteins. Using immunofluorescence and immunoelectron microscopy, we localized the Rab20 protein in the vicinity of the Golgi apparatus. Rab20 expression was detected by Western blot analysis in 11 of 11 pancreatic tumor cell lines and 7 of 8 primary pancreatic carcinomas. Absent or very faint expression was observed in normal pancreas cell extracts. Immunohistochemical analysis of Rab20 in tissues showed low or absent expression in normal pancreas and stronger expression in 15 of 18 exocrine pancreatic adenocarcinomas. Rab20 was also detected in preneoplastic pancreatic intraductal neoplasia lesions, suggesting that its up-regulation may be an early event in pancreatic carcinogenesis.

A Kruppel zinc finger of ZNF 146 interacts with the SUMO-1 conjugating enzyme UBC9 and is sumoylated in vivo.

The OZF (ZNF146) protein is a 33 kDa Kruppel protein, composed solely of 10 zinc finger motifs. It is overexpressed in the majority of pancreatic cancers and in more than 80% of colorectal cancers. We have identified OZF interacting factors with a yeast two-hybrid screen. Half of the positive clones characterized encoded UBC9, the E2 enzyme involved in the covalent conjugation of the small ubiquitin-like modifier 1 (SUMO-1). SUMO-1 is a 17 kDa migrating protein that is conjugated to several proteins and has been reported to exhibit multiple effects, including modulation of protein stability, subcellular localization, and gene expression. In HeLa cells transfected with OZF and SUMO-1 expression vectors, immunoblot revealed a major band migrating at 50 kDa and a minor band at 67 kDa, corresponding to the attachment to OZF of one and two SUMO-1 proteins, respectively. The relative amount of the sumoylated proteins increased following transfection with a UBC9 expression vector. The presence of the sumoylated form in HeLa cells solely transfected by OZF indicates the physiological activity of the endogenous SUMO-1 conjugation pathway. Using deletion mutants, we showed that two SUMO-1 modification sites are located on the sixth zinc finger. Mutation of two lysine residues greatly reduced the amount of the sumoylated form of OZF though their surrounding sequences differ from the consensus sequence reported for most proteins modified by SUMO-1 conjugation. Despite the presence of the sixth zinc finger, an OZF mutant containing zinc fingers 1-6 was not modified by SUMO-1 and failed to interact with UBC9. Addition of zinc finger 7 restored SUMO-1 modification and UBC9 interaction and provides evidence that a region downstream of the target lysines is required for interaction with UBC9, in order to achieve SUMO-1 modification. This is the first report of in vivo conjugation of a SUMO-1 protein to a Kruppel zinc finger motif.

Zinc finger protein overexpressed in colon carcinoma interacts with the telomeric protein hRap1.

The OZF (ZNF146) protein is a 33 kDa Kruppel protein, composed solely of 10 zinc finger motifs. It is overexpressed in the majority of pancreatic cancers and in more than 80% of colorectal cancers. We found an interaction between OZF and the telomeric hRap1 protein with a yeast two-hybrid screen. hRap1 (TERF2IP) is an ortholog of the yeast telomeric protein, scRap1 originally identified as a regulator of telomere length. In HeLa cells, it interacts with TRF2, a telomere repeat binding factor whose inactivation causes a dysregulation of telomere length and structure. Immunoprecipitation with anti-hRap1 antibodies in conditions that allow the purification of proteins associated with hRap1, demonstrated that OZF binds to hRap1 in HeLa cells. Using deletion mutants, we mapped the interacting domain of each protein. The three zinc fingers at the C-terminus of OZF interact with a region of hRap1 located downstream of the coil domain. It involves a stretch of at least 25 amino acids at the C-terminus of hRap1 that interact with TRF2. This suggests that OZF overexpression in tumours may alter the balance between hRap1 and other telomeric proteins and therefore that OZF function may be linked to telomere regulation.

The zinc finger protein OZF (ZNF146) is overexpressed in colorectal cancer.

Overexpression of the OZF gene has previously been demonstrated in the majority of pancreatic cancers. However, because the stages of tumour progression in this disease are poorly defined, no conclusion could be drawn concerning the relationship between OZF overexpression and the course of tumour progression. In contrast, initiation and progression steps are well defined in colorectal cancer. Most colon cancers are believed to arise from polypoid adenomas as a result of the gradual accumulation of genetic alterations, allowing the study of genetic events in the early stages of neoplasia. Accordingly, we wanted to assess the frequency of OZF overexpression in this tumour type and the relationship between overexpression and disease stage. Twenty-five colon carcinoma specimens from different sites and at various stages were analysed by immunoblotting using a highly specific mouse monoclonal antibody. Each sample was compared with adjacent normal colonic mucosa. Complementary immunohistochemical analysis was also carried out on a commercially available tissue array to identify cells expressing OZF. Of the 25 tumours analysed by immunoblotting, 20 expressed higher levels of OZF protein than their adjacent normal mucosa. Immunohistochemistry revealed OZF expression in tumour cells of 56/59 carcinomas and occasionally in infiltrating lymphocytes but at lower levels. Little or no staining was observed in cells taken from normal or inflammatory colon specimens. In both immunoblot and immunohistochemistry experiments, no correlation was found between OZF expression and clinical parameters such as TNM classification, stage, localization and age. Immunostaining was observed in low-grade adenomas, indicating that OZF expression occurs very early in the course of tumour progression. OZF expression, infrequent or absent in normal colonic mucosa, is present in more than 80% of colorectal cancers. Dysregulation of the OZF gene is an early event that may be implicated in the genesis of colonic carcinoma and may therefore provide a potential therapeutic target.

Targeted expression of the only zinc finger gene in transgenic mice is associated with impaired mammary development.

The only zinc finger (OZF) gene encodes a protein consisting mainly of 10 zinc finger motifs of the Krüppel type of yet unknown function. To potentially assess its in vivo role, mammary targeted deregulation of the expression of the murine gene was performed in transgenic mice using a goat beta-casein-based transgene. Mammary expression of the transgene was observed in the 11 lines obtained. In three expressing lines, this expression was tissue-specific and developmentally regulated. Further analysis of mice from two expressing lines revealed that transgene-homozygous females could not sustain full growth of their pups. This phenotype was associated with an impaired mammary gland development noticeable only after mid-gestation. It was characterised by an increase of the adipocyte to acini ratio and low or absence of fat globules within these acini compared to non-transgenic control animals. These transgenic observations strongly suggest that OZF is active in the mammary gland, interfering with the lactation process and thus that the described transgenic mice could be useful models to search for the cellular partner(s) of this protein.

Cloning of the mRNA of overexpression in colon carcinoma-1: a sequence overexpressed in a subset of colon carcinomas.

We have identified a novel human cDNA overexpressed in a colon carcinoma cell line, TC7, established from a tumor with a normal karyotype arising in a patient with a hereditary nonpolyposis colorectal carcinoma. The OCC-1 (overexpressed in colon carcinoma-1) gene is composed of six exons and located in the q24.1 region of chromosome 12. It is transcribed as two mRNA species that differ in their 5'- and 3'-terminal ends. Abundant accumulation of both transcripts was found in placenta, skeletal muscle, kidney, and pancreas tissues. Absent or very faint expression was observed in heart, brain, lung and liver tissues. Overexpressed in colon carcinoma-1 cDNA direct in vitro translation of several polypeptides whose size is shorter than 9 kDa. Attempts to produce antibodies against these synthesized polypeptides in Escherichia coli failed. The absence of sequences at the mRNA and DNA levels hybridizing with mouse sequences together with the absence of a large open reading frame raise the possibility that OCC-1 sequences appeared recently in evolution and are transcribed as two noncoding regulatory RNA. Elevated levels of OCC-1 mRNA were observed in three of eight colon carcinomas as compared to normal mucosa of the same patient. Since these tumors shared the same characteristics of having a near diploid karyotype, OCC-1 overexpression may be a hallmark of this subset of colon carcinomas.