BAMBI is a novel HIF1-dependent modulator of TGFß-mediated disruption of cell polarity during hypoxia.

Hypoxia and loss of cell polarity are common features of malignant carcinomas. Hypoxia-inducible factor 1 (HIF1) is the major regulator of cellular hypoxia response and mediates the activation of ∼300 genes. Increased HIF1 signaling is known to be associated with epithelial-mesenchymal transformation. Here, we report that hypoxia disrupts polarized epithelial morphogenesis of MDCK cells in a HIF1α-dependent manner by modulating the transforming growth factor-ß (TGFß) signaling pathway. Analysis of potential HIF1 targets in the TGFß pathway identified the bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI), a transmembrane glycoprotein related to the type I receptors of the TGFß family, whose expression was essentially lost in HIF1-depleted cells. Similar to what was observed in HIF1-deficient cells, BAMBI-depleted cells failed to efficiently activate TGFß signaling and retained epithelial polarity during hypoxia. Taken together, we show that hypoxic conditions promote TGFß signaling in a HIF1-dependent manner and BAMBI is identified in this pathway as a novel HIF1-regulated gene that contributes to hypoxia-induced loss of epithelial polarity.

Laminin 511 partners with laminin 332 to mediate directional migration of Madin-Darby canine kidney epithelial cells.

Sustained directional migration of epithelial cells is essential for regeneration of injured epithelia. Front-rear polarity of migrating cells is determined by local activation of a signaling network involving Cdc42 and other factors in response to spatial cues from the environment, the nature of which are obscure. We examined the roles of laminin (LM)-511 and LM-332, two structurally different laminin isoforms, in the migration of Madin-Darby canine kidney cells by suppressing expression of their α subunits using RNA interference. We determined that knockdown of LM-511 inhibits directional migration and destabilizes cell-cell contacts, in part by disturbing the localization and activity of the polarization machinery. Suppression of integrin α3, a laminin receptor subunit, in cells synthesizing normal amounts of both laminins has a similar effect as knockdown of LM-511. Surprisingly, simultaneous suppression of both laminin α5 and laminin α3 restores directional migration and cell-cell contact stability, suggesting that cells recognize a haptotactic gradient formed by a combination of laminins.

Molecular and cytological characterization of repetitive DNA sequences from the centromeric heterochromatin of Sciara coprophila.

Sciara coprophila (Diptera, Nematocera) constitutes a classic model to analyze unusual chromosome behavior such as the somatic elimination of paternal X chromosomes, the elimination of the whole paternal, plus non-disjunction of the maternal X chromosome at male meiosis. The molecular organization of the heterochromatin in S. coprophila is mostly unknown except for the ribosomal DNA located in the X chromosome pericentromeric heterochromatin. The characterization of the centromeric regions, thus, is an essential and required step for the establishment of S. coprophila as a model system to study fundamental mechanisms of chromosome segregation. To accomplish such a study, heterochromatic sections of the X chromosome centromeric region from salivary glands polytene chromosomes were microdissected and microcloned. Here, we report the identification and characterization of two tandem repeated DNA sequences from the pericentromeric region of the X chromosome, a pericentromeric RTE element and an AT-rich centromeric satellite. These sequences will be important tools for the cloning of S. coprophila centromeric heterochromatin using libraries of large genomic clones.

Autocrine transforming growth factor-{beta}1 activation mediated by integrin {alpha}V{beta}3 regulates transcriptional expression of laminin-332 in Madin-Darby canine kidney epithelial cells.

Laminin (LM)-332 is an extracellular matrix protein that plays a structural role in normal tissues and is also important in facilitating recovery of epithelia from injury. We have shown that expression of LM-332 is up-regulated during renal epithelial regeneration after ischemic injury, but the molecular signals that control expression are unknown. Here, we demonstrate that in Madin-Darby canine kidney (MDCK) epithelial cells LM-332 expression occurs only in subconfluent cultures and is turned-off after a polarized epithelium has formed. Addition of active transforming growth factor (TGF)-ß1 to confluent MDCK monolayers is sufficient to induce transcription of the LM α3 gene and LM-332 protein expression via the TGF-ß type I receptor (TßR-I) and the Smad2-Smad4 complex. Significantly, we show that expression of LM-332 in MDCK cells is an autocrine response to endogenous TGF-ß1 secretion and activation mediated by integrin αVß3 because neutralizing antibodies block LM-332 production in subconfluent cells. In confluent cells, latent TGF-ß1 is secreted apically, whereas TßR-I and integrin αVß3 are localized basolaterally. Disruption of the epithelial barrier by mechanical injury activates TGF-ß1, leading to LM-332 expression. Together, our data suggest a novel mechanism for triggering the production of LM-332 after epithelial injury.

Two new chromodomain-containing proteins that associate with heterochromatin in Sciara coprophila chromosomes.

We report here the molecular and cytological characterization of two proteins, ScoHET1 and ScoHET2 (for Sciara coprophila heterochromatin), which associate to constitutive heterochromatin in the dipteran S. coprophila. Both proteins, ScoHET1 of 37 kDa and ScoHET2 of 44 kDa, display two chromodomain motifs that contain the conserved residues essential for the recognition of methylated histone H3 at lysine 9. We raised antibodies to analyze the chromosomal location of ScoHET1 and ScoHET2 in somatic and germline cells. In S. coprophila polytene chromosomes, both proteins associate to the pericentromeric regions and to the heterochromatic subterminal bands of the chromosomes. In germinal nuclei, ScoHET1 and ScoHET2 proteins distribute to the heterochromatic regions of the regular chromosome complement and are abundantly present along the heterochromatic germline-limited "L" chromosomes. We investigated histone methylation modifications and found that all heterochromatic regions enriched in ScoHET1/ScoHET2 proteins exhibit high levels of di- and tri-methylated histone H3 at lysine 9. Taken together, our results support that the association of ScoHET1/ScoHET2 to heterochromatin is mediated by histone H3K9 methylation. Using 5-methylcytosine antibodies, we proved the cytological detection of DNA methylation in S. coprophila. From our observations in L germline chromosomes, heterochromatin in S. coprophila is highly enriched in DNA 5-methylcytosine residues.

Methylation of histone H3 at Lys4 differs between paternal and maternal chromosomes in Sciara ocellaris germline development.

An outstanding example of programmed chromosome elimination and genomic imprinting is found in sciarid flies (Diptera, Sciaridae), where whole chromosomes of paternal origin are selectively discarded from the genome during development. In early germ cells a single paternal X chromosome is eliminated in embryos of both sexes and in male meiotic cells the whole paternal complement is discarded. In sciarids, differential acetylation of histones H3 and H4 occurs between chromosomes of different parental origin, both in early germ nuclei and in male meiotic cells (Goday and Ruiz, 2002). We here investigated histone methylation modifications between chromosomes in germline cells of Sciara ocellaris. In early germ nuclei, maternal chromosomes show high levels of di- and trimethylated histone H3 at Lys4, whereas this histone modification is not detected in paternal chromosomes. In male meiosis, only the eliminated paternal chromosomes exhibit high levels of di- and trimethylated histones H3 at Lys4 and dimethylated H4 at Lys20. In early germ nuclei, RNA polymerase II associates to maternally-derived chromosomes but lacks phosphorylation of the C-terminal domain on Ser2. We found that histone H3 methylation at Lys4 does not correlate with transcriptional activity in early Sciara germline nuclei. The results support the conclusion that specific covalent chromatin modifications are involved in the imprinted behaviour of germline chromosomes in Sciara.