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Simplet-dependent regulation of ?-catenin signaling influences skeletal patterning downstream of Cx43.

ABSTRACT: The correct positioning of joints in the vertebrate skeleton is not well understood. Mutations in connexin43 (cx43) cause the short segment phenotype of the zebrafish short fin (sof^b123 ) mutant. We have shown that Cx43 suppresses evx1 expression, a transcription factor required for joint formation. Here, we provide novel insights into how Cx43 influences evx1 transcription. First, we find that Simplet (Smp) knockdown recapitulates the sof^b123 phenotypes of reduced regenerate length and reduced segment length, and we find evidence for synergy between cx43 and smp Moreover, knockdown of Smp increases the evx1 expression, similar to cx43 knockdown. Previous studies have shown that Smp is required for the nuclear localization of ?-catenin. Indeed, ?-catenin activity is required for segment length, and is reduced in both sof^b123 mutants and following Smp knockdown in regenerating fins. We further show that blocking canonical Wnt signaling results in a synergistic reduction in segment length in sof^b123 ^/+ heterozygotes. Together, our findings suggest that both Smp and ?-catenin function in a common molecular pathway with cx43 to influence both evx1 expression and joint location.

SUBMITTER: Bhattacharya S

PROVIDER: S-EPMC6288381 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Simplet-dependent regulation of β-catenin signaling influences skeletal patterning downstream of Cx43.

Bhattacharya Shashwati S Gargiulo Domenic D Iovine M Kathryn MK

Development (Cambridge, England) 20181130 23

The correct positioning of joints in the vertebrate skeleton is not well understood. Mutations in connexin43 (cx43) cause the short segment phenotype of the zebrafish short fin (sofb123 ) mutant. We have shown that Cx43 suppresses evx1 expression, a transcription factor required for joint formation. Here, we provide novel insights into how Cx43 influences evx1 transcription. First, we find that Simplet (Smp) knockdown recapitulates the sof< ...[more]

PMID: 30377172

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Project description:BACKGROUND:Aberrant Wnt/?-catenin signaling is associated with breast cancer even though genetic mutations in Wnt signaling components are rare. We have previously demonstrated that Rad6B, an ubiquitin conjugating enzyme, stabilizes ?-catenin via polyubiqutin modifications that render ?-catenin insensitive to proteasomal degradation. Rad6B is a transcriptional target of ?-catenin, creating a positive feedback loop between Rad6B expression and ?-catenin activation. METHODS:To isolate subpopulations expressing high or low Rad6B levels, we transfected MDA-MB-231 or WS-15 human breast cancer cells with ZsGreen fluorescent reporter vector in which the expression of ZsGreen was placed under the control of Rad6B promoter. ZsGreenhigh and ZsGreenlow subpopulations, reflective of high and low Rad6B promoter activity, respectively, were isolated by FACS. To determine the relevance of Wnt signaling in Rad6B-mediated ?-catenin stabilization/activation, the ZsGreenhigh cells were transfected with signaling-defective Wnt coreceptor LRP6?173. Rad6B expression and promoter activity were determined by RT-PCR, Western blot and Rad6B promoter-mediated luciferase assays. ?-catenin levels and transcriptional activity were determined by Western blot and TOP/FOP Flash reporter assays. Tumor formation and morphologies of ZsGreenlow, ZsGreenhigh, and ZsGreenhigh/LRP6?173 cells compared to unsorted vector controls were evaluated in nude mice. Expression of Wnt signaling related genes was profiled using the Wnt signaling pathway RT2 Profiler PCR arrays. RESULTS:ZsGreenhigh subpopulations showed high Rad6B expression and Rad6B promoter activity as compared to ZsGreenlow cells. ZsGreenhigh (high Rad6B expressors) also showed elevated ?-catenin levels and TOP/Flash activity. Inhibiting Wnt signaling in the high Rad6B expressors decreased ZsGreen fluorescence, Rad6B gene expression, ?-catenin levels and TOP/Flash activity. Tumors derived from high Rad6B expressors were predominantly composed of cells with epithelial mesenchymal transition (EMT) phenotype as compared to control tumors that were composed of both cuboidal and EMT-type cells. Tumors derived from low Rad6B expressors lacked EMT phenotype. Inhibition of LRP6 function in the high Rad6B expressors abrogated the EMT phenotype. Gene expression profiling showed upregulation of several Wnt signaling pathway regulators in high Rad6B expressors that were downregulated by interference of Wnt signaling with mutant LRP6 or by Rad6B silencing. CONCLUSIONS:These data reveal a functional link between the canonical Wnt pathway and Rad6B in ?-catenin activation and breast cancer progression.

Dataset Information

Simplet-dependent regulation of ?-catenin signaling influences skeletal patterning downstream of Cx43.

Publications

Simplet-dependent regulation of β-catenin signaling influences skeletal patterning downstream of Cx43.

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