ADAM13 knockdown in Xenopus laevis cranial neural crest
Ontology highlight
ABSTRACT:
ORGANISM(S): Xenopus laevis
PROVIDER: GSE21517 | GEO | 2011/02/22
SECONDARY ACCESSION(S): PRJNA125977
REPOSITORIES: GEO
Similar Datasets
Project description:ADAMs are transmembrane metalloproteases that control cell behavior by cleaving both cell adhesion and signaling molecules. The cytoplasmic domain of ADAMs can regulate the proteolytic activity by controlling the subcellular localization and/or the activation of the protease domain. Here we show that the cytoplasmic domain of ADAM13 is cleaved and translocates into the nucleus. Preventing this translocation renders the protein incapable of promoting cranial neural crest (CNC) cell migration in vivo, without affecting its proteolytic activity. In addition, the cytoplasmic domain of ADAM13 regulates the expression of multiple genes in the CNC. This study shows that the cytoplasmic domain of ADAM metalloproteases can perform essential functions in the nucleus of cells and may contribute substantially to the overall function of the protein. Total RNA from cranial neural crest explants dissected from Xenopus laevis embryos at stage 15 to 17. Embryos were injected with either control morpholino (CMO), morpholino to ADAM13 (MO13) or MO13 and a fusion between GFP- and the cytoplasmic domain of ADAM13 (GFP-C13). Triplicate microarray correspond to independent experiments labeled DA4, DA5 and DA2. For each sample 1 is CMO, sample 2 is MO13 and sample 3 is MO13+C13. Embryos were injected with 10 ng of morpholino and 0.5 ng of GFP-C13 mRNA. Supplementary files: [1] matrix2_sig-genes.txt CNC array: Genes significantly affected by the loss of ADAM13 p<0.01. Normalized probe set generated by GC-RMA. [2] matrix3_sig-genes.txt CNC array: Genes significantly affected by the loss of ADAM13 and the addition of GFP-C13 p<0.01. Normalized probe set generated by GC-RMA.
2011-02-22 | E-GEOD-21517 | biostudies-arrayexpress
Project description:Full-length membrane PTK7 and its N-terminal and C-terminal proteolytic fragments induced differential transcriptional profiles in HT1080 cells. Pseudokinase PTK7 is an essential regulator of planar cell polarity (PCP) and directional cell motility in the course of vertebrate development and embryogenesis, and regulates both the non-canonical Wnt/PCP and canonical Wnt pathways. In contrast to its well-appreciated, crucial role in embryo development, the functional importance of the intact full-length PTK7 in malignancy is still a matter of debate. In humans, the full-length membrane PTK7 consists of seven extracellular immunoglobulin-like (Ig) domains, a transmembrane region, a juxtamembrane region and a catalytically inert cytoplasmic tyrosine kinase (PTK) domain. Because pericellular proteolysis plays a primary role in cell migration, especially in the directional locomotion of tumor cells, it is likely that proteolysis and PCP converge to promote efficient directed cancer cell migration. In agreement, the functionality of PTK7 is directly regulated by proteolysis. Ubiquitous membrane type-1 matrix metalloproteinase (MT1-MMP), arguably the primary enzyme in pericellular proteolysis and cancer cell migration, cleaves the PKP621M-bM-^FM-^SLI sequence in the seventh Ig-like domain of membrane PTK7 and this cleavage results in the liberation of the N-terminal soluble PTK7 fragment (sPTK7). MT1-MMP proteolysis is followed by the cleavage of the C-terminal residual portion of PTK7 by ADAMs, including ADAM17. The ectodomain shedding is a prerequisite for the intramembrane cleavage of PTK7 by M-NM-3-secretase. This cleavage releases the C-terminal cytoplasmic tail fragment of PTK7, which is then either degraded by the proteasome or transported to the nucleus.The limited pre-existing data suggest that the full-length membrane PTK7 and its proteolytic products cause an opposing effect on the efficiency of cell migration. Thus, the continuing presence of the full-length membrane PTK7 on the plasma membrane down-regulated the myosin light chain (MLC) phosphorylation (a downstream event of the Wnt/PCP pathway) and, in agreement, reduced migration efficiency of fibrosarcoma HT1080 cells. MT1-MMP proteolysis of PTK7 reversed the inhibitory effect of the full-length membrane PTK7, resulted in the accumulation of the stable N-terminal sPTK7 fragment in the extracellular milieu and promoted cell invasion of HT1080 cells. Expression of the Chz PTK7 mutant that exhibited an additional PEKM-bM-^FM-^SLK503 MT1-MMP cleavage site in the junction region between the fifth and the sixth Ig-like domains stimulated cell migration even further. These effects suggest the existence of the intriguing and specific downstream mechanisms by which the intact PTK7, its digest fragments and the homo- and heterodimeric complexes between the PTK7 membrane, soluble and intracellular portions control cell function. These mechanisms, however, have not been precisely investigated in the earlier works by us and others. Understanding of these mechanisms will shed additional light on the role that PTK7 alone as well as in its combination with MT1-MMP, ADAMs and M-NM-3-secretase plays in cancer cell migration. To evaluate in detail the effects of PTK7 and its proteolytic fragments on genome-wide transcriptional regulation, we specifically employed fibrosarcoma HT1080 cells. These highly invasive cells express low endogenous levels of PTK7 but high levels of active MT1-MMP and ADAMs. Because of these parameters, we could manipulate this preudokinase functionality using HT1080 cells transfected with the recombinant PTK7 constructs. The cells we employed included the PTK7 knock-out cells (shPTK7 cells), cells with the enforced overexpression of the original membrane PTK7 (PTK7 cells) and its Chuzhoi (Chz) mutant that exhibited an additional PEKM-bM-^FM-^SLK503 MT1-MMP cleavage site (Chz cells), and the cells, which overexpresssed multiple deletion species of PTK7. These species represented the soluble (sPTK7), membrane and cytoplasmic C-terminal digest fragments that resulted because of PTK7 cleavage by MT1-MMP (cPTK7/622-1070) and ADAM/gammaM-bM-^@M-^Ssecretase (cPTK7/726-1070). Expression of these constructs allowed us to specifically determine the downstream effect of PTK7 proteolysis on gene expression. HT1080 cells were stably transfected with the PTK7 constructs. Cells (1 x 10^4/ml) were plated in DMEM- 10% FBS in a 100-mm dish and grown for 72 h to produce a subconfluent culture. Total cellular RNA was extracted using a Direct-zol RNA MiniPrep kit (Zymo Research). Biotin-labeled cRNA samples were prepared using the Illumina RNA Amplification Kit (Ambion). The labeled cRNA (750 ng) was hybridized for 18 h at 58M-BM-0C to the HumanHT-12 v4 Expression BeadChip (Illumina). BeadChips were then developed using fluorolink streptavidin-Cy3 (GE Healthcare). Array chips were scanned using an Illumina BeadArray Reader. The initial data extraction and normalization were performed using the BeadArray Reader and GeneSpring GX software (Agilent).
2013-12-16 | E-GEOD-53340 | biostudies-arrayexpress
Project description:Craniofacial development depends on cell-cell interactions, coordinated cellular movement and differentiation under the control of regulatory gene networks, which include the distal-less (Dlx) gene family. However, the functional significance of Dlx5 in patterning the oropharyngeal region has remained unknown. Here we show that loss of Dlx5 leads to a shortened soft palate and an absence of the levator veli palatini, palatopharyngeus, and palatoglossus muscles that are derived from the 4th pharyngeal arch (PA), but the tensor veli palatini, derived from the 1st PA, is unaffected. Dlx5-positive cranial neural crest (CNC) cells are in direct contact with myoblasts derived from the pharyngeal mesoderm, and Dlx5 disruption leads to altered proliferation and apoptosis of CNC and muscle progenitor cells. Moreover, the FGF10 pathway is downregulated in Dlx5-/- mice, and activation of FGF10 signaling rescues CNC cell proliferation and myogenic differentiation in these mutant mice. Collectively, our results indicate that Dlx5 plays critical roles in patterning of the oropharyngeal region and development of muscles derived from the 4th PA mesoderm in the soft palate, likely via interactions between CNC-derived and myogenic progenitor cells.
2017-10-15 | GSE104163 | GEO
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Project description:Full-length membrane PTK7 and its N-terminal and C-terminal proteolytic fragments induced differential transcriptional profiles in HT1080 cells. Pseudokinase PTK7 is an essential regulator of planar cell polarity (PCP) and directional cell motility in the course of vertebrate development and embryogenesis, and regulates both the non-canonical Wnt/PCP and canonical Wnt pathways. In contrast to its well-appreciated, crucial role in embryo development, the functional importance of the intact full-length PTK7 in malignancy is still a matter of debate. In humans, the full-length membrane PTK7 consists of seven extracellular immunoglobulin-like (Ig) domains, a transmembrane region, a juxtamembrane region and a catalytically inert cytoplasmic tyrosine kinase (PTK) domain. Because pericellular proteolysis plays a primary role in cell migration, especially in the directional locomotion of tumor cells, it is likely that proteolysis and PCP converge to promote efficient directed cancer cell migration. In agreement, the functionality of PTK7 is directly regulated by proteolysis. Ubiquitous membrane type-1 matrix metalloproteinase (MT1-MMP), arguably the primary enzyme in pericellular proteolysis and cancer cell migration, cleaves the PKP621↓LI sequence in the seventh Ig-like domain of membrane PTK7 and this cleavage results in the liberation of the N-terminal soluble PTK7 fragment (sPTK7). MT1-MMP proteolysis is followed by the cleavage of the C-terminal residual portion of PTK7 by ADAMs, including ADAM17. The ectodomain shedding is a prerequisite for the intramembrane cleavage of PTK7 by γ-secretase. This cleavage releases the C-terminal cytoplasmic tail fragment of PTK7, which is then either degraded by the proteasome or transported to the nucleus.The limited pre-existing data suggest that the full-length membrane PTK7 and its proteolytic products cause an opposing effect on the efficiency of cell migration. Thus, the continuing presence of the full-length membrane PTK7 on the plasma membrane down-regulated the myosin light chain (MLC) phosphorylation (a downstream event of the Wnt/PCP pathway) and, in agreement, reduced migration efficiency of fibrosarcoma HT1080 cells. MT1-MMP proteolysis of PTK7 reversed the inhibitory effect of the full-length membrane PTK7, resulted in the accumulation of the stable N-terminal sPTK7 fragment in the extracellular milieu and promoted cell invasion of HT1080 cells. Expression of the Chz PTK7 mutant that exhibited an additional PEK↓LK503 MT1-MMP cleavage site in the junction region between the fifth and the sixth Ig-like domains stimulated cell migration even further. These effects suggest the existence of the intriguing and specific downstream mechanisms by which the intact PTK7, its digest fragments and the homo- and heterodimeric complexes between the PTK7 membrane, soluble and intracellular portions control cell function. These mechanisms, however, have not been precisely investigated in the earlier works by us and others. Understanding of these mechanisms will shed additional light on the role that PTK7 alone as well as in its combination with MT1-MMP, ADAMs and γ-secretase plays in cancer cell migration.
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