microscopy-based-methods-for-assessment-epithelial-cell-migration

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During  <em>In Vitro</em> Wound Healing

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated ...

IMIB-Arrixaca

In mammals, estrogens regulate the immune system, either directly or indirectly via several leukocyte types through autocrine/paracrine mechanisms. In the gilthead seabream (Sparus aurata L.) gonad, an intensive remodeling process accompanied by the massive infiltration of acidophilic granulocytes (AG) is partially triggered by 17β-estradiol (E(2)). Once AG infiltrated the gonad, show impaired activities. In this study we first demonstrate that neither testicular nor head-kidney AG express any of the three estrogen receptor (ER) genes (ERa, ERb1 and ERb2) described in the gilthead seabream, while head-kidney macrophages (Mc) and lymphocytes (Ly) constitutively express ERa gene. Moreover, Mc are important in the immune-modulatory role of E(2), as suggested by its ability to induce ERb2 gene expression and up-regulate the expression of genes coding for ERa, ERb1, pro-inflammatory cytokines, chemokines and tissue remodeling molecules. Furthermore, the soluble factors produced by E(2)-treated Mc decreased in head-kidney phagocytes, their phagocytic ability and capacity, while no effects were observed on their reactive oxygen intermediate (ROI) production or their migratory capabilities. However, the role of Ly in the regulation of AG migration and the modulation of phagocytic and ROI production activities triggered by E(2) can not be ruled out, so that further studies are necessary to clarify these issues.

17β-Estradiol regulates gilthead seabream professional phagocyte responses through macrophage activation.

Although several studies have demonstrated the ability of some endocrine disruptive chemicals (EDCs) to alter the physiology of zebrafish, the immune-reproductive interaction has received little attention in this species. In this study, we used a homozygous line carrying an insertion of 8 amino acids in the ligand-binding domain of the estrogen receptor 2b gene (esr2b) to further understand the role of estrogen signaling on innate immunity. Adult mutant fish showed distorted sexual ratios related with alterations in testicular morphology and supraphysiological testosterone and 17β-estradiol (E2) levels. Immunity-wise, although esr2b mutant fish showed unaltered antibacterial responses, they were unable to mount an effective antiviral response upon viral challenge. RT-qPCR analysis demonstrated that mutant fish were able to induce the genes encoding major antiviral molecules, including Ifnphi1, Ifnphi2, Infphi3, Mxb and Mxc, and the negative feedback regulator of cytokine signaling Socs1. Notably, although esr2b mutant larvae showed a similar resistance to SVCV infection to their wild type siblings, waterborne E2 increased their viral susceptibility. Similarly, the exposure of adult wild type zebrafish to E2 also resulted in increased susceptibility to SVCV infection. Finally, the administration of recombinant Ifnphi1 hardly reversed the higher viral susceptibility of esr2b mutant zebrafish, suggesting that elevated socs1 levels impair Ifn signaling. All together, these results uncover an important role for E2 and Esr signaling in the fine-tuning of sexual hormone balance and the antiviral response of vertebrates.

Estrogen receptor 2b deficiency impairs the antiviral response of zebrafish.

The importance of histamine in the physiology of the testis in mammals and reptiles has been recently shown. Histamine receptors (Hrs) are well conserved in fish and are functional in several fish species. We report here for the first time that histamine and the mRNA of Hrh1, Hrh2 and Hrh3 are all present in the gonad of the hermaphrodite teleost fish gilthead seabream. Moreover, cimetidine, which acts in vitro as an agonist of Hrh1 and Hrh2 on this species, was intraperitoneally injected in one and two years old gilthead seabream males. After three and five days of cimetidine injection, we found that this compound differently modified the gonadal hrs transcript levels and affects the testicular cell renewal and the gene expression of steroidogenesis-related molecules as well as the serum steroid levels. Our data point to cimetidine as a reproductive disruptor and elucidate a role for histamine in the gonad of this hermaphrodite fish species through Hr signalling.

Cimetidine disrupts the renewal of testicular cells and the steroidogenesis in a hermaphrodite fish.

Keratinocyte migration is a mandatory aspect of wound healing. We have previously shown that amniotic membrane (AM) applied to chronic wounds assists healing through a process resulting in the overexpression of c-Jun at the wound's leading edge. We have also demonstrated that AM modifies the genetic programme induced by transforming growth factor-ß (TGF-ß) in chronic wounds. Here we used a scratch assay of mink lung epithelial cells (Mv1Lu) and a spontaneously immortalized human keratinocyte cell line (HaCaT) cells to examine the influence of AM application on the underlying signalling during scratch closure. AM application induced c-Jun phosphorylation at the leading edge of scratch wounds in a process dependent on MAPK and JNK signalling. Strikingly, when the TGF-ß-dependent Smad-activation inhibitor SB431542 was used together with AM, migration improvement was partially restrained, whereas the addition of TGF-ß had a synergistic effect on the AM-induced cell migration. Moreover, antagonizing TGF-ß with specific antibodies in both cell lines or knocking out TGF-ß receptors in Mv1Lu cells had similar effects on cell migration as using SB431542. Furthermore, we found that AM was able to attenuate TGF-ß-Smad signalling specifically at the migrating edge; AM treatment abated Smad2 and Smad3 nuclear localization in response to TGF-ß in a process dependent on mitogen-activated protein kinase kinase 1 (MEK1) activation but independent of EGF receptor or JNK activation. The involvement of Smad signalling on AM effects on HaCaT keratinocytes was further corroborated by overexpression of either Smad2 or Smad3 and the use of Smad phosphorylation-specific inhibitors, revealing a differential influence on AM-induced migration for each Smad. Thus, AM TGF-ß-Smad signalling abating is essential for optimal cell migration and wound closure.

Amniotic membrane stimulates cell migration by modulating transforming growth factor-β signalling.

During wound healing, the migration of keratinocytes onto newly restored extracellular matrix aims to reestablish continuity of the epidermis. The application of amniotic membrane (AM) to chronic, deep traumatic, non-healing wounds has proven successful at stimulating re-epithelialization. When applied on epithelial cell cultures, AM activates extracellular signal-regulated kinases 1/2 (ERK1/2) and c-Jun N-terminal kinases 1/2 (JNK1/2), with the overexpression and phosphorylation of c-Jun along the wound edge. The effect of AM on the migration of cells was investigated by studying critical proteins involved in the focal adhesions turn-over: Focal Adhesion Kinase (FAK), Paxillin and Vinculin. In Mv1Lu and HaCaT cells, validated models for cell migration and wound healing, AM affected the expression and activation of Paxillin, but did not affect Vinculin expression, both factors which integrate into focal adhesions. Moreover, AM regulation also affected FAK activity through phosphorylation. Finally, we have determined that AM regulation of focal adhesions involves both JNK and MEK MAP kinase signaling pathways. This data provides a molecular background to understand how AM regulates critical cell and molecular aspects of cell migration, organizing and directing the movement of cells by the continuous formation, maturation, and turnover of focal adhesion structures at the migration leading edge.

Sergio Liarte

Laboratorio de Oncología Molecular y TGF-β

IMIB-Arrixaca

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During In Vitro Wound Healing

Sergio Liarte

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Laboratorio de Oncología Molecular y TGF-β

IMIB-Arrixaca

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During In Vitro Wound Healing

Laboratorio de Oncología Molecular y TGF-β