The beneficial role of extracellular reactive oxygen species in apoptosis-induced compensatory proliferation

Apoptosis-induced proliferation (AiP) maintains tissue homeostasis following massive stress-induced cell death. During this phenomenon, dying cells induce proliferation of the surviving cells to compensate for the tissue loss, and thus restore organ size. Along with wound healing and tissue regeneration, AiP also contributes to tumor repopulation following radiation or chemotherapy. There are several models of AiP. Using an “undead” AiP model that causes hyperplastic overgrowth of Drosophila epithelial tissue, we recently demonstrated that extracellular reactive oxygen species (eROS) are produced by undead epithelial cells, and are necessary for inducing AiP and overgrowth. Furthermore, hemocytes, the Drosophila blood cells, are seen adjacent to the undead epithelial tissue, and may secrete the TNF ortholog Eiger that signals through the TNF receptor to active Jun-N-terminal kinase (JNK) in the undead tissue and induce proliferation. We propose that undead epithelial tissue triggers an inflammatory response that resembles recruitment of macrophages to human epithelial tumors, and that these tumor-associated macrophages release signals for proliferation and tumor growth of the epithelium. This Extra View article summarizes these recent findings with a focus on the role of eROS for promoting regeneration and inflammation-induced tumorigenesis.     

CA125/MUC16 interacts with Src family kinases,and over-expression of its C-terminal fragment in human epithelial cancer cells reduces cell–cell adhesion

MUC16/CA125 is over-expressed in human epithelial tumors including ovarian, breast and some other carcinomas. The purpose of this study is to investigate how cell surface MUC16 is functionally involved in tumor progression, with a special focus on the role of its cytoplasmic tail. Forced expression of C-terminal MUC16 fragment (MUC16C) in epithelial cancer cells increased cell migration. We found that MUC16C directly interacted with Src family kinases (SFKs). Notably, localizations of E-cadherin and β-catenin at the cell–cell contacts were more diffuse in MUC16C transfectants compared with mock transfectants. Furthermore, MUC16C transfectants showed reduced Ca²⁺-dependent cell–cell adhesion, but the treatment of cells with PP2, a SFKs inhibitor, restored this. Because cell surface MUC16 is also associated with the E-cadherin/β-catenin complex, the over-expression of MUC16 and its interaction with SFKs may enhance SFKs-induced deregulation of E-cadherin. Thus, our results suggest a role for cell surface MUC16 in cell–cell adhesion of epithelial cancer cells.     

Epithelial argininosuccinate synthetase is dispensable for intestinal regeneration and tumorigenesis

The epithelial signaling pathways involved in damage and regeneration, and neoplastic transformation are known to be similar. We noted upregulation of argininosuccinate synthetase (ASS1) in hyperproliferative intestinal epithelium. Since ASS1 leads to de novo synthesis of arginine, an important amino acid for the growth of intestinal epithelial cells, its upregulation can contribute to epithelial proliferation necessary to be sustained during oncogenic transformation and regeneration. Here we investigated the function of ASS1 in the gut epithelium during tissue regeneration and tumorigenesis, using intestinal epithelial conditional Ass1 knockout mice and organoids, and tissue specimens from colorectal cancer patients. We demonstrate that ASS1 is strongly expressed in the regenerating and Apc-mutated intestinal epithelium. Furthermore, we observe an arrest in amino acid flux of the urea cycle, which leads to an accumulation of intracellular arginine. However, loss of epithelial Ass1 does not lead to a reduction in proliferation or increase in apoptosis in vivo, also in mice fed an arginine-free diet. Epithelial loss of Ass1 seems to be compensated by altered arginine metabolism in other cell types and the liver.Subject terms: Cancer metabolism, Regeneration, Cancer metabolism, Experimental models of disease, Oncogenesis     

Expression profiles of elastase1 (NvElastaseI) and secretory leukocyte protease inhibitor (NvSLPI) during forelimb regeneration in adult Notophthalmus viridescens suggest a role in epithelial remodeling and delamination

Extracellular proteases and their inhibitors may regulate a number of important processes involved in forelimb regeneration in the adult newt, including epithelial remodeling, breakdown of extracellular matrix, and dedifferentiation. We have identified a newt homologue of human ElastaseI (NvElastaseI) and its potential inhibitor, SLPI (NvSLPI), and evaluated their spatial and temporal expression during limb regeneration. NvElastaseI is upregulated early in regeneration and is associated with subdermal and wound epithelial cells, suggesting an involvement in wound healing and the generation of the wound epithelium. Up until 15 days post-amputation, NvElastaseI is also scattered throughout the developing blastema and may have a role in the dedifferentiation of stump tissues. NvSLPI is found at the interface between the intact skin and the wound epithelium, and may limit NvElastaseI activity. NvSLPI is also expressed in dermal glands, and is likely involved in anti-microbial activity or function. Quite apart from regeneration, complementary patterns of expression of NvElastaseI and NvSLPI are associated with newt epithelial sloughing.     

C-terminal Src kinase-homologous kinase (CHK), a unique inhibitor inactivating multiple active conformations of Src family tyrosine kinases

The Src family of protein kinases (SFKs) mediates mitogenic signal transduction, and constitutive SFK activation is associated with tumorigenesis. To prevent constitutive SFK activation, the catalytic activity of SFKs in normal mammalian cells is suppressed mainly by two inhibitors called C-terminal Src kinase (CSK) and CSK-homologous kinase (CHK), which inactivate SFKs by phosphorylating a consensus tyrosine near the C terminus of SFKs (Y(T)). The phosphorylated Y(T) intramolecularly binds to the SH2 domain of SFKs. This interaction, known as pY(T)/SH2 interaction, together with binding between the SH2 kinase linker and the SH3 domain of SFKs (linker/SH3 interaction) stabilizes SFKs in a "closed" inactive conformation. We previously discovered an alternative mechanism CHK employs to inhibit SFKs. This mechanism, referred to as the non-catalytic inhibitory mechanism, involves tight binding of CHK to SFKs; the binding alone is sufficient to inhibit SFKs. Herein, we constructed multiple active conformations of an SFK member, Hck, by systematically disrupting the two inhibitory interactions. We found that CHK employs the non-catalytic mechanism to inactivate these active conformations of Hck. However, CHK does not bind Hck when it adopts the inactive conformation in which both inhibitory interactions are intact. These data indicate that binding of CHK to SFKs via the non-catalytic mechanism is governed by the conformations of SFKs. Although CSK is also an inhibitor of SFKs, it does not inhibit SFKs by a similar non-catalytic mechanism. Thus, the non-catalytic inhibitory mechanism is a unique property of CHK that allows it to down-regulate multiple active conformations of SFKs.     

Autophagy regulator BECN1 suppresses mammary tumorigenesis driven by WNT1 activation and following parity

Earlier studies reported allelic deletion of the essential autophagy regulator BECN1 in breast cancers implicating BECN1 loss, and likely defective autophagy, in tumorigenesis. Recent studies have questioned the tumor suppressive role of autophagy, as autophagy-related gene (Atg) defects generally suppress tumorigenesis in well-characterized mouse tumor models. We now report that, while it delays or does not alter mammary tumorigenesis driven by Palb2 loss or ERBB2 and PyMT overexpression, monoallelic Becn1 loss promotes mammary tumor development in 2 specific contexts, namely following parity and in association with wingless-type MMTV integration site family, member 1 (WNT1) activation. Our studies demonstrate that Becn1 heterozygosity, which results in immature mammary epithelial cell expansion and aberrant TNFRSF11A/TNR11/RANK (tumor necrosis factor receptor superfamily, member 11a, NFKB activator) signaling, promotes mammary tumorigenesis in multiparous FVB/N mice and in cooperation with the progenitor cell-transforming WNT1 oncogene. Similar to our Becn1^+/?;MMTV-Wnt1 mouse model, low BECN1 expression and an activated WNT pathway gene signature correlate with the triple-negative subtype, TNFRSF11A axis activation and poor prognosis in human breast cancers. Our results suggest that BECN1 may have nonautophagy-related roles in mammary development, provide insight in the seemingly paradoxical roles of BECN1 in tumorigenesis, and constitute the basis for further studies on the pathophysiology and treatment of clinically aggressive triple negative breast cancers (TNBCs).     

src family kinases regulate renal epithelial paracellular permeability barrier through an occludin‐independent mechanism

Paracellular permeability is mediated by the epithelial cell tight junction. Studies in intestinal and other epithelia have suggested that the activity of src family kinases (SFKs) increases epithelial paracellular permeability through its action on the tight junction protein, occludin, but the involvement of SFKs and occludin in regulation of renal epithelial paracellular permeability is unclear. In this study, the role of SFKs in regulation of renal epithelial paracellular permeability and the involvement of occludin protein in this regulatory event was examined in two renal epithelial cell lines, LLC‐PK₁ (proximal tubule‐like) and MDCK (distal tubule‐like). The effect of broad spectrum SFK inhibitors on paracellular permeability of calcein and fluorescein‐dextran3000 were examined. SFK inhibitor treatment increased paracellular movement of both compounds in both renal epithelial cell lines. The SFK inhibitor effect was concentration‐dependent and, at low concentrations, was not associated with cell damage/death. Response to SFK inhibitors was acquired progressively after cell populations attained confluence suggesting maturation of the regulatory mechanism. Increased paracellular permeability was not associated with dramatic changes in total cell content of occludin protein, its partitioning between detergent‐soluble and ‐insoluble fractions, or its subcellular localization. Further, the SFK‐induced increase in paracellular permeability was unaffected by either occludin protein overexpression or occludin protein knockdown. These results demonstrate that SFK activity decreases paracellular permeability of renal epithelial cells, as opposed to its effect in intestinal epithelial cells, and that this regulation is not mediated by occludin protein. J. Cell. Physiol. 228: 1210–1220, 2013. © 2012 Wiley Periodicals, Inc.     

Early redox,Src family kinase,and calcium signaling integrate wound responses and tissue regeneration in zebrafish

Tissue injury can lead to scar formation or tissue regeneration. How regenerative animals sense initial tissue injury and transform wound signals into regenerative growth is an unresolved question. Previously, we found that the Src family kinase (SFK) Lyn functions as a redox sensor in leukocytes that detects H₂O₂ at wounds in zebrafish larvae. In this paper, using zebrafish larval tail fins as a model, we find that wounding rapidly activated SFK and calcium signaling in epithelia. The immediate SFK and calcium signaling in epithelia was important for late epimorphic regeneration of amputated fins. Wound-induced activation of SFKs in epithelia was dependent on injury-generated H₂O₂. A SFK member, Fynb, was responsible for fin regeneration. This work provides a new link between early wound responses and late regeneration and suggests that redox, SFK, and calcium signaling are immediate “wound signals” that integrate early wound responses and late epimorphic regeneration.     

Signalling through Src family kinase isoforms is not redundant in models of thrombo‐inflammatory vascular disease

The Src family kinases (SFK) are a group of signalling molecules with important regulatory functions in inflammation and haemostasis. Leucocytes and platelets express multiple isoforms of the SFKs. Previous studies used broad‐spectrum pharmacological inhibitors, or murine models deficient in multiple SFK isoforms, to demonstrate the functional consequences of deficiencies in SFK signalling. Here, we hypothesized that individual SFK operate in a non‐redundant fashion in the thrombo‐inflammatory recruitment of monocyte during atherosclerosis. Using in vitro adhesion assays and single SFK knockout mice crossed with the ApoE^?/? model of atherosclerosis, we find that SFK signalling regulates platelet‐dependent recruitment of monocytes. However, loss of a single SFK, Fgr or Lyn, reduced platelet‐mediated monocyte recruitment in vitro. This translated into a significant reduction in the burden of atherosclerotic disease in Fgr^?/?/ApoE^?/? or Lyn^?/?/ApoE^?/? animals. SFK signalling is not redundant in thrombo‐inflammatory vascular disease and individual SFK may represent targets for therapeutic intervention.     

Purinergic agonists stimulate lens Na-K-ATPase-mediated transport via a Src tyrosine kinase-dependent pathway

The Na-K-ATPase is vital for maintenance of lens transparency. Past studies using intact lens suggested the involvement of tyrosine kinases in short-term regulation of Na-K-ATPase. Furthermore, in vitro phosphorylation of a lens epithelial membrane preparation by Src family kinases (SFKs), a family of nonreceptor tyrosine kinases, resulted in modification of Na-K-ATPase activity. Here, the effect of purinergic agonists, ATP and UTP, on Na-K-ATPase function and SFK activation was examined in the rabbit lens. Na-K-ATPase function was examined using two different approaches, measurement of ouabain-sensitive potassium (⁸⁶Rb) uptake by the intact lens, and Na-K-ATPase activity in lens epithelial homogenates. ATP and UTP caused a significant increase in ouabain-sensitive potassium (⁸⁶Rb) uptake. Na-K-ATPase activity was increased in the epithelium of lenses pretreated with ATP. Lenses treated with ATP or UTP displayed activation of SFKs as evidenced by increased Western blot band density of active SFK (phosphorylated at the active loop Y416) and decreased band density of inactive SFKs (phosphorylated at the COOH terminal). A single PY416-Src immunoreactive band at 60 kDa was observed, suggesting not all Src family members are activated. Immunoprecipitation studies showed that band density of active Src, and to a lesser extent active Fyn, was significantly increased, while active Yes did not change. Preincubation of the lenses with SFK inhibitor PP2 abolished the ATP-induced increase in ouabain-sensitive potassium (⁸⁶Rb) uptake. The results suggest selective activation of Src and/or Fyn is part of a signaling mechanism initiated by purinergic agonists that increases Na-K-ATPase-mediated transport in the organ-cultured lens. Src kinase; receptors     

Transforming Potential of Src Family Kinases Is Limited by the Cholesterol-Enriched Membrane Microdomain

The upregulation of Src family kinases (SFKs) has been implicated in cancer progression, but the molecular mechanisms regulating their transforming potentials remain unclear. Here we show that the transforming ability of all SFK members is suppressed by being distributed to the cholesterol-enriched membrane microdomain. All SFKs could induce cell transformation when overexpressed in C-terminal Src kinase (Csk)-deficient fibroblasts. However, their transforming abilities varied depending on their affinity for the microdomain. c-Src and Blk, with a weak affinity for the microdomain due to a single myristate modification at the N terminus, could efficiently induce cell transformation, whereas SFKs with both myristate and palmitate modifications were preferentially distributed to the microdomain and required higher doses of protein expression to induce transformation. In contrast, disruption of the microdomain by depleting cholesterol could induce a robust transformation in Csk-deficient fibroblasts in which only a limited amount of activated SFKs was expressed. Conversely, the addition of cholesterol or recruitment of activated SFKs to the microdomain via a transmembrane adaptor, Cbp/PAG1, efficiently suppressed SFK-induced cell transformation. These findings suggest that the membrane microdomain spatially limits the transforming potential of SFKs by sequestering them away from the transforming pathways.Src family kinases (SFKs) are membrane-associated, non-receptor protein tyrosine kinases involved in a variety of intracellular signaling pathways (5). SFKs are comprised of eight members in mammals: c-Src, Fyn, c-Yes, Lyn, Lck, Hck, c-Fgr and Blk. Among these, c-Src, Fyn, and c-Yes are ubiquitously expressed, whereas the others are relatively concentrated in hematopoietic cell lineages. The intracellular distribution of each SFK also varies depending on their unique N-terminal sequences and acyl modifications (5, 27). These distinctive features of SFKs suggest that each SFK member plays a unique role in particular tissues or cells. In contrast, it is also known that SFKs have redundant and pleiotropic functions in regulating critical cellular events, such as cell division, motility, adhesion, angiogenesis, and survival (26). In a variety of human cancers, protein levels and/or specific activities of c-Src and c-Yes are frequently upregulated (13, 35). Upregulation of Lyn, Lck, Hck, c-Fgr, or Blk is also observed in some leukemias and lymphomas (10, 16, 26). These observations imply a role for SFKs in cell transformation, tumorigenesis, and metastasis (31). However, because SFK genes are rarely mutated in human cancers (31), the mechanisms underlying their upregulation in these cancers remain unclear. Furthermore, the distinctive expression patterns and functional redundancy among SFK members have hampered concurrent analyses of their intrinsic transforming abilities and contribution to cancer progression.In normal cells, the kinase activity of SFKs is negatively regulated by the phosphorylation of its C-terminal regulatory Tyr residue by C-terminal Src kinase (Csk) (21, 22). The cytoplasmic Csk requires Csk-binding scaffold proteins to gain efficient access to membrane-bound SFKs. Previously, we identified a transmembrane adaptor protein, Cbp (also known as PAG1), as a specific Csk-binding protein. Cbp/PAG1 is exclusively localized to a membrane microdomain enriched by cholesterol and sphingolipids and plays a scaffolding role for Cbp/PAG1 in Csk-mediated negative regulation of SFKs (3, 15). We also reported that expression of Cbp/PAG1 is noticeably downregulated by c-Src transformation and in some human cancer cells and that reexpression of Cbp/PAG1 can suppress c-Src-induced transformation and tumorigenesis (23). In addition, we showed that Cbp/PAG1 suppressed c-Src function independently of Csk by directly sequestering activated c-Src in the membrane microdomain. These findings suggest a potential role for Cbp/PAG1 as a suppressor for c-Src-mediated cancer progression. However, whether Cbp/PAG1 would serve as a suppressor for other SFK members and whether other microdomain adaptors, such as LIME (4, 11), would also contribute to the suppression of SFK-mediated transformation have yet to be examined.The membrane microdomain has been regarded as a signaling platform that harbors various signaling molecules and positively transduces cell signaling evoked by activated receptors (29). This model has been best exemplified in immunoreceptor-mediated signaling (8). Moreover, it was reported that SFKs could function positively when bound to Cbp/PAG1 in the microdomain (30, 32). Such positive roles of the microdomain in cell signaling are apparently inconsistent with its suppressive role in Src-mediated transformation. However, this discrepancy rather raises the possibility that the membrane microdomain would function to segregate or protect the normal signaling pathway from the transforming pathways. To prove this hypothesis, more extensive analysis of the role of the membrane microdomain in controlling cell transformation remains to be performed (28).To elucidate the role of the membrane microdomain in regulating the functions of SFKs, we first compared the transforming abilities of all SFK members using Csk-deficient cells, a reconstitution system in which wild-type SFKs can induce cell transformation (24), and we evaluated the relevance of the membrane distribution of SFKs to their transforming activities. We then investigated the role of the microdomain by disrupting or enhancing its function using methyl-β-cyclodextrin (MβCD) and a microdomain-specific adaptor, Cbp/PAG1, respectively. Our results show that the membrane microdomain and Cbp/PAG1 spatially limit the oncogenic potential of SFKs by sequestering them away from the transforming pathways.     

Upregulation of matrix metalloproteinase triggers transdifferentiation of retinal pigmented epithelial cells in Xenopus laevis: A Link between inflammatory response and regeneration

In adult Xenopus eyes, when the whole retina is removed, retinal pigmented epithelial (RPE) cells become activated to be retinal stem cells and regenerate the whole retina. In the present study, using a tissue culture model, it was examined whether upregulation of matrix metalloproteinases (Mmps) triggers retinal regeneration. Soon after retinal removal, Xmmp9 and Xmmp18 were strongly upregulated in the tissues of the RPE and the choroid. In the culture, Mmp expression in the RPE cells corresponded with their migration from the choroid. A potent MMP inhibitor, 1,10‐PNTL, suppressed RPE cell migration, proliferation, and formation of an epithelial structure in vitro. The mechanism involved in upregulation of Mmps was further investigated. After retinal removal, inflammatory cytokine genes, IL‐1β and TNF‐α , were upregulated both in vivo and in vitro. When the inflammation inhibitors dexamethasone or Withaferin A were applied in vitro, RPE cell migration was severely affected, suppressing transdifferentiation. These results demonstrate that Mmps play a pivotal role in retinal regeneration, and suggest that inflammatory cytokines trigger Mmp upregulation, indicating a direct link between the inflammatory reaction and retinal regeneration. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1086–1100, 2017     

A genetic approach to in vitro regeneration of non-regenerating cotton (Gossypium hirsutum L.) cultivars

Selections were made among individual plants of Gossypium hirsutum cv `Coker 310' for high-frequency in vitro regeneration by somatic embryogenesis. After three generations of selection, a pure line for high-frequency somatic embryogenesis was selected and named Coker 310 FR (FR, fully regenerating). Coker 310 FR could be regenerated by following previously published protocols (see Materials and methods) and a modified protocol developed in this study that reduced the time necessary for in vitro regeneration. Coker 310 FR was crossed with individual plants of major cotton cultivars grown in India, namely `MCU 5', `MCU 7', `Khandwa 2', `Bikaneri Nerma', `F 846' that have been shown to be recalcitrant to in vitro regeneration, to evaluate the regeneration potential of F₁s. All the F₁s showed regeneration by somatic embryogenesis. However, the F₁ of G. barbadense×G. hirsutum Coker 310 FR did not regenerate. Received: 16 September 1997 / Revision received: 1 April 1998 / Accepted: 15 May 1998     

Monolayer formation and DNA synthesis of the outer epithelial cells from pearl oyster mantle in coculture with amebocytes

Summary In vitro experiments were conducted to clarify the involvement of the epithelium-amebocyte interaction in epithelial regeneration of bivalves. The outer epithelia of the pallial mantle of the pearl oyster, Pinctada fucata martensii, were separated in cell sheets from the inner connective tissue layers by digestion with Dispase. Clumps of the separated mantle epithelia were inoculated onto the amebocyte layers prepared on the bottom of culture dishes and maintained at 20° C in 5% CO₂:95% air for 1 wk. Balanced salt solution with 0.03% (wt/vol) glucose was used as a culture medium. The epithelial cells adhered to the amebocyte layers within 24 h, changed their shape from cuboidal to squamous, and migrated and formed monolayer sheets within 3 d. Electron microscopy confirmed maintenance of epithelial polarity and cell to cell junction in the sheets; 6 d after the inoculation, 5-bromo-2′-deoxyuridine was added to the culture at 30 μM. After labeling for 24 h, the cultures were fixed and stained with anti 5-bromo-2′-deoxyuridine antibody. Cells with immunoreactive nuclei were clearly observed in the epithelial cell sheets, indicating active DNA synthesis in the epithelial sheets. Thus, cocultured with amebocytes, the outer epithelial cells from pallial mantle tissue formed a monolayer sheet and started DNA synthesis. The morphological features of the mantle outer epithelial cells are analogous to those described for the in vivo cutaneous wound healing process, suggesting that the epithelium-amebocyte interaction is important in the regeneration of epithelium in bivalves.     

Snai1 regulates cell lineage allocation and stem cell maintenance in the mouse intestinal epithelium

Snail family members regulate epithelial‐to‐mesenchymal transition (EMT) during invasion of intestinal tumours, but their role in normal intestinal homeostasis is unknown. Studies in breast and skin epithelia indicate that Snail proteins promote an undifferentiated state. Here, we demonstrate that conditional knockout of Snai1 in the intestinal epithelium results in apoptotic loss of crypt base columnar stem cells and bias towards differentiation of secretory lineages. In vitro organoid cultures derived from Snai1 conditional knockout mice also undergo apoptosis when Snai1 is deleted. Conversely, ectopic expression of Snai1 in the intestinal epithelium in vivo results in the expansion of the crypt base columnar cell pool and a decrease in secretory enteroendocrine and Paneth cells. Following conditional deletion of Snai1, the intestinal epithelium fails to produce a proliferative response following radiation‐induced damage indicating a fundamental requirement for Snai1 in epithelial regeneration. These results demonstrate that Snai1 is required for regulation of lineage choice, maintenance of CBC stem cells and regeneration of the intestinal epithelium following damage.