Abstract
Background: Previously published work has demonstrated that the LPS injection of Ciona robusta leads to the overexpression of a truncated form of an immune-related mRNA (C8short) by means of Ciona robusta (CR) alternative polyadenylation (APA) (CR-APA). Methods: The 3D structure of the C8short-derived Ciona robusta chemo-attractive peptide (CrCP) was evaluated by homology modeling. The biological activity of the CrCP was studied in vitro using a primary human dermal cell line (HuDe). Real-Time PCR was used to investigate the expression levels of genes involved in cell motility. NF-κB signaling was studied by western blotting. Results: In silico modeling showed that CrCP displayed structural characteristics already reported for a short domain of the vertebrate CRK gene, suggesting its possible involvement in cell migration mechanisms. In vitro assays demonstrated that CrCP was capable of inducing the motility of HuDe cells in both wound healing and chemo-attractive experiments. qPCR demonstrated the capability of CrCP to modulate the expression of the matrix metalloproteinase-7 (MMP-7) and E-cadherin genes. Finally, western blot analysis demonstrated that treatment with CrCP induced activation of the NF-κB signaling pathway. Conclusion: Our results describe the characterization of the 3D structure and chemo-attractive activity of an LPS-induced CrCP peptide from Ciona robusta.
Highlights
Tunicates are the closest relatives to vertebrates [1,2] and, like other invertebrates, their immune response relies exclusively on innate immunity
We examined its 3D structure by means of in silico modeling, which showed a certain degree of homology to a protein domain of the human cancer-related signaling adaptor protein CT10 Regulator of Kinase (CRK) [31], a vertebrate gene that has been demonstrated to induce cytoskeletal reorganization during cell migration
In a previously published paper, we demonstrated that chemo-attractive peptide (CrCP) did not show any cytotoxic or hemolytic effects on either immortalized or primary human cell lines within a large range of concentrations and up to 48 hours of incubation [33]
Summary
Tunicates are the closest relatives to vertebrates [1,2] and, like other invertebrates, their immune response relies exclusively on innate immunity. The ascidian Ciona robusta (formerly Ciona intestinalis type A) [4] was the first invertebrate chordate to have its genome sequenced, allowing for the analysis of developmental mechanisms, genome-wide gene regulatory networks, epigenetic regulatory mechanisms and gene expression profiles at single-cell resolution [5,6,7,8] In this scenario, Ciona robusta has been extensively utilized to study inflammatory response after challenge with foreign agents, such as the injection. Drugs 2020, 18, 209 of erythrocytes [9,10], proteins [11] and LPS [12] into the tunic and, in particular, into the pharynx, which is considered the immunocompetent organ [13] These stimuli can induce a complex cascade of events, leading to the activation of immune-related gene pathways and to the recruitment of numerous hemocytes infiltrating the tunic tissue, where they can release several inflammation mediators [14]. Western blot analysis demonstrated that treatment with CrCP induced activation of the NF-κB signaling pathway
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