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Pan-cyclin-dependent-kinase (CDK) inhibitors are a new class of targeted therapies that can act on multiple CDKs, with dinaciclib being one of the most promising compounds. Although used as a monotherapy, an interesting approach could be to combine it with radiotherapy. Here, we show that dinaciclib increases radiosensitivity in some experimental models of lung and colon cancer (A549 or HCT 116) but not in others (H1299 or HT-29). Dinaciclib did not alter serine-protein kinase ATM signalling or cell cycle profiling after ionising-radiation exposure, which have been described for other CDK inhibitors. Interestingly, in terms of apoptosis, although the combination renders a clear increase, no potentiation of the ionising-radiation-induced apoptosis was observed. Mechanistically, inhibition of CDK12 by dinaciclib diminishes BRCA1 expression, which decreases homologous recombination (HR) and probably promotes the nonhomologous end joining repair process (NHEJ), which ultimately promotes the induction of ionising-radiation-associated cellular senescence in a TP53-dependent manner, explaining the lack of effect observed in some experimental models. In conclusion, our report proposes a molecular mechanism, based on the signalling axis CDK12-BRCA1, involved in this newly identified therapeutic effect of dinaciclib, although other players implicated in HR should not be discarded. In addition, our data provide a rationale for more selective and personalised chemo/radiotherapy treatment according to the genetic background of the tumour., (© 2024 The Author(s). Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

In the treatment of hormone-dependent cancers, aromatase inhibitors (AI) are receiving increased attention due to some undesirable effects such as the risk of endometrial cancer and thromboembolism of SERMs (selective estrogen receptor modulators). Letrozole is the most active AI with 99% aromatase inhibition. Unfortunately, this compound also exhibits some adverse effects such as hot flashes and fibromyalgias. Therefore, there is an urgent need to explore new types of AIs that retain the same-or even increased-antitumor ability. Inspired by the letrozole structure, a set of new derivatives has been synthesized that include a ferrocenyl moiety and different heterocycles. The derivative that contains a benzimidazole ring, namely compound 6, exhibits a higher aromatase inhibitory activity than letrozole and it also shows potent cytostatic behavior when compared to other well-established aromatase inhibitors, as demonstrated by dose-response, cell cycle, apoptosis and time course experiments. Furthermore, 6 promotes the inhibition of cell growth in both an aromatase-dependent and -independent fashion, as indicated by the study of A549 and MCF7 cell lines. Molecular docking and molecular dynamics calculations on the interaction of 6 or letrozole with the aromatase binding site revealed that the ferrocene moiety increases the van der Waals and hydrophobic interactions, thus resulting in an increase in binding affinity. Furthermore, the iron atom of the ferrocene fragment can form a metal-acceptor interaction with a propionate fragment, and this results in a stronger coupling with the heme group-a possibility that is consistent with the strong aromatase inhibition of 6., (© 2023. The Author(s), under exclusive licence to Society for Biological Inorganic Chemistry (SBIC).)

Background and Purpose: MAPKs are among the most relevant signalling pathways involved in coordinating cell responses to different stimuli. This group includes p38MAPKs, constituted by 4 different proteins with a high sequence homology: MAPK14 (p38α), MAPK11 (p38β), MAPK12 (p38γ) and MAPK13 (p38δ). Despite their high similarity, each member shows unique expression patterns and even exclusive functions. Thus, analysing protein-specific functions of MAPK members is necessary to unequivocally uncover the roles of this signalling pathway. Here, we investigate the possible role of MAPK11 in the cell response to ionizing radiation (IR)., Materials and Methods: We developed MAPK11/14 knockdown through shRNA and CRISPR interference gene perturbation approaches and analysed the downstream effects on cell responses to ionizing radiation in A549, HCT-116 and MCF-7 cancer cell lines. Specifically, we assessed IR toxicity by clonogenic assays; DNA damage response activity by immunocytochemistry; apoptosis and cell cycle by flow cytometry (Annexin V and propidium iodide, respectively); DNA repair by comet assay; and senescence induction by both X-Gal staining and gene expression of senescence-associated genes by RT-qPCR., Results: Our findings demonstrate a critical role of MAPK11 in the cellular response to IR by controlling the associated senescent phenotype, and without observable effects on DNA damage response, apoptosis, cell cycle or DNA damage repair., Conclusion: Our results highlight MAPK11 as a novel mediator of the cellular response to ionizing radiation through the control exerted onto IR-associated senescence., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Author(s).)

Over the last 30 years, the study of the cellular response to ionizing radiation (IR) has increased exponentially. Among the various signaling pathways affected by IR, p38 MAPK has been shown to be activated both in vitro and in vivo, with involvement in key processes triggered by IR-mediated genotoxic insult, such as the cell cycle, apoptosis or senescence. However, we do not yet have a definitive clue about the role of p38 MAPK in terms of radioresistance/sensitivity and its potential use to improve current radiotherapy. In this review, we summarize the current knowledge on this family of MAPKs in response to IR as well as in different aspects related to radiotherapy, such as their role in the control of REDOX, fibrosis, and in the radiosensitizing effect of several compounds.

Sarcomas are a heterogeneous group of tumors in which the role of ERK5 is poorly studied. To clarify the role of this MAPK in sarcomatous pathology, we used a murine 3-methyl-cholanthrene (3MC)-induced sarcoma model. Our data show that 3MC induces pleomorphic sarcomas with muscle differentiation, showing an increased expression of ERK5. Indeed, this upregulation was also observed in human sarcomas of muscular origin, such as leiomyosarcoma or rhabdomyosarcoma. Moreover, in cell lines derived from these 3MC-induced tumors, abrogation of Mapk7 expression by using specific shRNAs decreased in vitro growth and colony-forming capacity and led to a marked loss of tumor growth in vivo. In fact, transcriptomic profiling in ERK5 abrogated cell lines by RNAseq showed a deregulated gene expression pattern for key biological processes such as angiogenesis, migration, motility, etc., correlating with a better prognostic in human pathology. Finally, among the various differentially expressed genes, Klf2 is a key mediator of the biological effects of ERK5 as indicated by its specific interference, demonstrating that the ERK5-KLF2 axis is an important determinant of sarcoma biology that should be further studied in human pathology.

Sorafenib is a multikinase inhibitor widely used in cancer therapy with an antitumour effect related to biological processes as proliferation, migration or invasion, among others. Initially designed as a Raf inhibitor, Sorafenib was later shown to also block key molecules in tumour progression such as VEGFR and PDGFR. In addition, sorafenib has been connected with key signalling pathways in cancer such as EGFR/EGF. However, no definitive clue about the molecular mechanism linking sorafenib and EGF signalling pathway has been established so far. Our data in HeLa, U2OS, A549 and HEK293T cells, based on in silico, chemical and genetic approaches demonstrate that the MEK5/ERK5 signalling pathway is a novel target of sorafenib. In addition, our data show how sorafenib is able to block MEK5-dependent phosphorylation of ERK5 in the Ser218/Tyr220, affecting the transcriptional activation associated with ERK5. Moreover, we demonstrate that some of the effects of this kinase inhibitor onto EGF biological responses, such as progression through cell cycle or migration, are mediated through the effect exerted onto ERK5 signalling pathway. Therefore, our observations describe a novel target of sorafenib, the ERK5 signalling pathway, and establish new mechanistic insights for the antitumour effect of this multikinase inhibitor., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

The problems of resistance and side effects associated with cisplatin and other chemotherapeutic drugs have boosted research aimed at finding new compounds with improved properties. The use of platinum(IV) prodrugs is one alternative, although there is some controversy regarding the predictive ability of the peak reduction potentials. In the work described here a series of fourteen chloride Pt(II) and Pt(IV) compounds was synthesised and fully characterised. The compounds contain different bidentate arylazole heterocyclic ligands. Their cytotoxic properties against human lung carcinoma (A549), human breast carcinoma (MCF7) and human colon carcinoma (HCT116 and HT29) cell lines were studied. A clear relationship between the type of ligand and the anti-proliferative properties was found, with the best results obtained for the Pt(II) compound that contains an aniline fragment, (13), thus evidencing a positive effect of the NH 2 group. Stability and aquation studies in DMSO, DMF and DMSO/water mixtures were carried out on the active complexes and an in-depth analysis of the two aquation processes, including DFT analysis, of 13 was undertaken. It was verified that DNA was the target and that cell death occurred by apoptosis in the case of 13. Furthermore, the cytotoxic derivatives did not exhibit haemolytic activity. The reduction of the Pt(IV) compounds whose Pt(II) congeners were active was studied by several techniques. It was concluded that the peak reduction potential was not useful to predict the ability for reduction. However, a correlation between the cytotoxic activity and the standard reduction potential was found., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background and Purpose: Gemcitabine is an antitumour agent currently used in the treatment of several types of cancer with known properties as a radiosensitizer. p38MAPK signalling pathway has been shown to be a major determinant in the cellular response to gemcitabine in different experimental models. However, the molecular mechanism implicated in gemcitabine-associated radiosensitivity remains unknown., Materials and Methods: The human sarcoma cell lines A673 and HT1080, and a mouse cell line derived from a 3-methylcholanthrene induced sarcoma were used as experimental models. Modulation of p38MAPKs was performed by pharmacological approaches (SB203580) and genetic interference using lentiviral vectors coding for specific shRNAs. Viability was assessed by MTT. Gene expression was evaluated by western blot and RT-qPCR. Induction of apoptosis was monitored by caspase 3/7 activity. Response to ionizing radiation was evaluated by clonogenic assays., Results: Our data demonstrate that chemical inhibition of p38MAPK signalling pathway blocks gemcitabine radiosensitizing potential. Genetic interference of MAPK14 (p38α), the most abundantly expressed and best characterized p38MAPK, despite promoting resistance to gemcitabine, it does not affect its radiosensitizing potential. Interestingly, specific knockdown of MAPK11 (p38β) induces a total loss of the radiosensitivity associated to gemcitabine, as well as a marked increase in the resistance to the drug., Conclusion: The present work identifies p38β as a major determinant of the radiosensitizing potential of gemcitabine without implication of p38α, suggesting that p38β status should be analysed in those cases in which gemcitabine is combined with ionizing radiation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The p38 mitogen-activated protein kinase (MAPK) signaling pathway is implicated in cancer biology and has been widely studied over the past two decades as a potential therapeutic target. Most of the biological and pathological implications of p38MAPK signaling are often associated with p38α (MAPK14). Recently, several members of the p38 family, including p38γ and p38δ, have been shown to play a crucial role in several pathologies including cancer. However, the specific role of p38β (MAPK11) in cancer is still elusive, and further investigation is needed. Here, we summarize what is currently known about the role of p38β in different types of tumors and its putative implication in cancer therapy. All evidence suggests that p38β might be a key player in cancer development, and could be an important therapeutic target in several pathologies, including cancer.

Purpose: Autophagy has lately emerged as an important biological process with implications in several hematological pathologies. Recently, a growing body of evidence supports a putative role of autophagy in chronic lymphocytic leukemia; however, no definitive clue has been established so far. To elucidate this issue, we have developed a pilot study to measure autophagic flux in peripheral blood mononuclear cells from chronic lymphocytic leukemia patients, and explored its correlation with classical clinical/analytical parameters., Methods/patients: Thirty-three chronic lymphocytic leukemia patients participated in the study. Autophagic flux in peripheral blood mononuclear cells was determined by western blot measuring the levels of the proteins p62 and lipidated LC3. Moreover, p62 mRNA levels were analyzed by RT-qPCR., Results: Lymphocytosis and the percentage of tumoral lymphocytes in chronic lymphocytic leukemia patients statistically correlate with a blocked autophagic flux., Conclusion: Alterations in autophagic flux could play an important role in the physiopathology of chronic lymphocytic leukemia.

Targeting cell cycle has become one of the major challenges in cancer therapy, being Palbociclib, a CDK4/6 inhibitor, an excellent example. Recently, it has been reported that Palbociclib could be a novel radiosensitizer agent. In an attempt to clarify the molecular basis of this effect we have used cell lines from colorectal (HT29, HCT116) lung (A549, H1299) and breast cancer (MCF-7). Our results indicate that the presence of a p53 wild type is strictly required for Palbociclib to exert its radiosensitizing effect, independently of the inhibitory effect exerted on CDK4/6. In fact, abrogation of p53 in cells with functional p53 blocks the radiosensitizing effect of Palbociclib. Moreover, no radiosensitizing effect is observed in cells with non-functional p53, but restoration of p53 function promotes radiosensitivity associated to Palbociclib. Furthermore, the presence of Palbociclib blocks the transcriptional activity of p53 in an ATM-dependent-fashion after ionizing radiation exposure, as the blockage of p21/WAF1 expression demonstrates. These observations are a proof of concept for a more selective therapy, based on the combination of CDK4/6 inhibition and radiotherapy, which would only benefit to those patients with a functional p53 pathway., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Objectives: To fully clarify the role of Mitogen Activated Protein Kinase in the therapeutic response to Sorafenib in Renal Cell Carcinoma as well as the cell death mechanism associated to this kinase inhibitor, we have evaluated the implication of several Mitogen Activated Protein Kinases in Renal Cell Carcinoma-derived cell lines., Materials and Methods: An experimental model of Renal Cell Carcinoma-derived cell lines (ACHN and 786-O cells) was evaluated in terms of viability by MTT assay, induction of apoptosis by caspase 3/7 activity, autophagy induction by LC3 lipidation, and p62 degradation and kinase activity using phospho-targeted antibodies. Knock down of ATG5 and ERK5 was performed using lentiviral vector coding specific shRNA., Results: Our data discard Extracellular Regulated Kinase 1/2 and 5 as well as p38 Mitogen Activated Protein Kinase pathways as mediators of Sorafenib toxic effect but instead indicate that the inhibitory effect is exerted through the PI3K/Akt signalling pathway. Furthermore, we demonstrate that inhibition of Akt mediates cell death associated to Sorafenib without caspase activation, and this is consistent with the induction of autophagy, as indicated by the use of pharmacological and genetic approaches., Conclusion: The present report demonstrates that Sorafenib exerts its toxic effect through the induction of autophagy in an Akt-dependent fashion without the implication of Mitogen Activated Protein Kinase. Therefore, our data discard the use of inhibitors of the RAF-MEK-ERK1/2 signalling pathway in RCC and support the use of pro-autophagic compounds, opening new therapeutic opportunities for Renal Cell Carcinoma., Competing Interests: The authors have declared that no competing interests exist in relation with this work.

The E1a gene from adenovirus has become a major tool in cancer research. Since the discovery of E1a, it has been proposed to be an oncogene, becoming a key element in the model of cooperation between oncogenes. However, E1a's in vivo behaviour is consistent with a tumour suppressor gene, due to the block/delay observed in different xenograft models. To clarify this interesting controversy, we have evaluated the effect of the E1a 13s isoform from adenovirus 5 in vivo. Initially, a conventional xenograft approach was performed using previously unreported HCT116 and B16-F10 cells, showing a clear anti-tumour effect regardless of the mouse's immunological background (immunosuppressed/immunocompetent). Next, we engineered a transgenic mouse model in which inducible E1a 13s expression was under the control of cytokeratin 5 to avoid side effects during embryonic development. Our results show that E1a is able to block chemical skin carcinogenesis, showing an anti-tumour effect. The present report demonstrates the in vivo anti-tumour effect of E1a, showing that the in vitro oncogenic role of E1a cannot be extrapolated in vivo, supporting its future use in gene therapy approaches., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The involvement of NOTCH signaling in macrophage activation by Toll receptors has been clearly established, but the factors and pathways controlling NOTCH signaling during this process have not been completely delineated yet. We have characterized the role of TSPAN33, a tetraspanin implicated in a disintegrin and metalloproteinase (ADAM) 10 maturation, during macrophage proinflammatory activation. Tspan33 expression increases in response to TLR signaling, including responses triggered by TLR4, TLR3, and TLR2 activation, and it is enhanced by IFN-γ. In this study, we report that induction of Tspan33 expression by TLR and IFN-γ is largely dependent on NOTCH signaling, as its expression is clearly diminished in macrophages lacking Notch1 and Notch2 expression, but it is enhanced after overexpression of a constitutively active intracellular domain of NOTCH1. TSPAN33 is the member of the TspanC8 tetraspanin subgroup more intensely induced during macrophage activation, and its overexpression increases ADAM10, but not ADAM17, maturation. TSPAN33 favors NOTCH processing at the membrane by modulating ADAM10 and/or Presenilin1 activity, thus increasing NOTCH signaling in activated macrophages. Moreover, TSPAN33 modulates TLR-induced proinflammatory gene expression, at least in part, by increasing NF-κB-dependent transcriptional activity. Our results suggest that TSPAN33 represents a new control element in the development of inflammation by macrophages that could constitute a potential therapeutic target., (Copyright © 2016 by The American Association of Immunologists, Inc.)

DLK1 (PREF1, pG2, or FA1) is a transmembrane and secreted protein containing epidermal growth factor-like repeats. Dlk1 expression is abundant in many tissues during embryonic and fetal development and is believed to play an important role in the regulation of tissue differentiation and fetal growth. After birth, Dlk1 expression is abolished in most tissues but is possibly reactivated to regulate stem cell activation and responses to injury. We have recently reported that DLK1 regulates many aspects of salivary gland organogenesis. Here, we have extended our studies of the salivary gland phenotype of Dlk1 knock-out mice. We have observed that salivary glands are smaller and weigh significantly less in both Dlk1 knock-out males and females compared with gender and age-matched wild-type mice and regardless of the natural sexual dimorphism in rodent salivary glands. This reduced size correlates with a reduced capacity of Dlk1-deficient mice to secrete saliva after stimulation with pilocarpine. However, histological and ultrastructural analyses of both adult and developing salivary gland tissues have revealed no defects in Dlk1 ((-/-)) mice, indicating that genetic compensation accounts for the relatively mild salivary phenotype in these animals. Finally, despite their lack of severe anomalies, we have found that salivary glands from Dlk1-deficient mice present a higher amount of CK14-positive epithelial progenitors at various developmental stages, suggesting a role for DLK1 in the regulation of salivary epithelial stem cell balance.

Delta-like protein 1 (DLK1) is a noncanonical ligand that inhibits NOTCH1 receptor activity and regulates multiple differentiation processes. In macrophages, NOTCH signaling increases TLR-induced expression of key pro-inflammatory mediators. We have investigated the role of DLK1 in macrophage activation and inflammation using Dlk1-deficient mice and Raw 264.7 cells overexpressing Dlk1. In the absence of Dlk1, NOTCH1 expression is increased and the activation of macrophages with TLR3 or TLR4 agonists leads to higher production of IFN-β and other pro-inflammatory cytokines, including TNF-α, IL-12, and IL-23. The expression of key proteins involved in IFN-β signaling, such as IRF3, IRF7, IRF1, or STAT1, as well as cRel, or RelB, which are responsible for the generation of IL-12 and IL-23, is enhanced in Dlk1 KO macrophages. Consistently, Dlk1 KO mice are more sensitive to LPS-induced endotoxic shock. These effects seem to be mediated through the modulation of NOTCH1 signaling. TLR4 activation reduces DLK1 expression, whereas increases NOTCH1 levels. In addition, DLK1 expression diminishes during differentiation of human U937 cells to macrophages. Overall, these results reveal a novel role for DLK1 as a regulator of NOTCH-mediated, pro-inflammatory macrophage activation, which could help to ensure a baseline level preventing constant tissue inflammation., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Resistance to cisplatin is a major challenge in the current cancer therapy. In order to explore new therapeutic strategies to cisplatin resistance, we evaluated, in a model of lung cancer (H1299 and H460 cell lines), the nature of the pathways leading to cell death. We observed that H1299 displayed a natural resistance to cisplatin due to an inability to trigger an apoptotic response that correlates with the induction of autophagy. However, pharmacological and genetic approaches showed how autophagy was a mechanism associated to cell death rather than to resistance. Indeed, pro-autophagic stimuli such as mTOR or Akt inhibition mediate cell death in both cell lines to a similar extent. We next evaluated the response to a novel platinum compound, monoplatin, able to promote cell death in an exclusive autophagy-dependent manner. In this case, no differences were observed between both cell lines. Furthermore, in response to monoplatin, two molecular hallmarks of cisplatin response (p53 and MAPKs) were not implicated, indicating the ability of this pro-autophagic compound to overcome cisplatin resistance. In summary, our data highlight how induction of autophagy could be used in cisplatin resistant tumours and an alternative treatment for p53 mutated patient in a synthetic lethally approach.

Aims: The epidermal growth factor-like protein Delta-like 1 (DLK1) regulates multiple differentiation processes. It resembles NOTCH ligands structurally and is considered a non-canonical ligand. Given the crucial role of the NOTCH pathway in angiogenesis, we hypothesized that DLK1 could regulate angiogenesis by interfering with NOTCH. We therefore investigated the expression and function of DLK1 in the vascular endothelium and its role in the regulation of angiogenesis., Methods and Results: We report DLK1 expression in the endothelium of different species, including human, cow, pig, and mouse. Angiogenesis was studied by using in vitro and in vivo models of angiotube formation in endothelial cells, retinal phenotypes in Dlk1-null mice, and vessel development in zebrafish. DLK1 overexpression strongly inhibited angiotube formation, whereas lung endothelial cells from Dlk1-null mice were highly angiogenic. In vivo studies demonstrated DLK1-mediated inhibition of neovessel formation and revealed an altered pattern of angiogenesis in the retinas of Dlk1-null mice. The expression of human DLK1 in zebrafish embryos severely altered the formation of intersegmental vessels, while knockdown of the orthologous gene was associated with ectopic and increased tumour-induced angiogenesis. NOTCH-dependent signalling as determined by gene expression reporters was inhibited by the presence of DLK1 in vascular endothelial cells. In contrast, Dlk1-null mice showed increased levels of NOTCH downstream targets, such as Snail and Slug., Conclusion: Our results unveil a novel inhibitory role for DLK1 in the regulation of angiogenesis, mediated by antagonism of the NOTCH pathway, and establish the basis for investigating its action in pathological settings.

To better understand the role of the non-canonical Notch ligand delta-like protein 1 (DLK1), in hormone-producing cells, we studied the cell distribution and subcellular localisation of DLK1 in the pituitary of male adult 129/SvJ mice, and analysed the variations in the hormone-producing cells associated with the lack of this gene in Dlk1 knockout mice. The results obtained showed the presence of DLK1-immunoreactive (ir) cells in all hormone-producing cells of the anterior pituitary. Immunoelectron microscopy showed DLK1-ir in the rough endoplasmic reticulum and inside secretory vesicles, suggesting that DLK1 is released together with pituitary hormones. Moreover, we found that prolactin (PRL)-DLK1-ir cells are in intimate contact with follicle-stimulating hormone (FSH)-ir-DLK1-negative cells. In Dlk1 knockout mice, we detected a significantly lower number of gowth hormone (GH)-ir cells, a reduction in the FSH and PRL immunostaining intensity, and a significant decrease in FSH mRNA expression compared to wild-type mice. An increase in pituitary GH mRNA expression and serum leptin levels was also found. These findings provide evidence supporting several regulatory functions of DLK1 in the pituitary gland., (© 2011 The Authors. Journal of Neuroendocrinology © 2011 Blackwell Publishing Ltd.)

Delta-like 1/fetal antigen 1 (DLK1/FA-1) is a transmembrane protein belonging to the Notch/Delta family that acts as a membrane-associated or a soluble protein to regulate regeneration of a number of adult tissues. Here we examined the role of DLK1/FA-1 in bone biology using osteoblast-specific Dlk1-overexpressing mice (Col1-Dlk1). Col1-Dlk1 mice displayed growth retardation and significantly reduced total body weight and bone mineral density (BMD). Micro-computed tomographis (µCT) scanning revealed a reduced trabecular and cortical bone volume fraction. Tissue-level histomorphometric analysis demonstrated decreased bone-formation rate and enhanced bone resorption in Col1-Dlk1 mice compared with wild-type mice. At a cellular level, Dlk1 markedly reduced the total number of bone marrow (BM)-derived colony-forming units fibroblasts (CFU-Fs), as well as their osteogenic capacity. In a number of in vitro culture systems, Dlk1 stimulated osteoclastogenesis indirectly through osteoblast-dependent increased production of proinflammatory bone-resorbing cytokines (eg, Il7, Tnfa, and Ccl3). We found that ovariectomy (ovx)-induced bone loss was associated with increased production of Dlk1 in the bone marrow by activated T cells. Interestingly, Dlk1(-/-) mice were significantly protected from ovx-induced bone loss compared with wild-type mice. Thus we identified Dlk1 as a novel regulator of bone mass that functions to inhibit bone formation and to stimulate bone resorption. Increasing DLK1 production by T cells under estrogen deficiency suggests its possible use as a therapeutic target for preventing postmenopausal bone loss., (Copyright © 2011 American Society for Bone and Mineral Research.)

The protein DLK2, highly homologous to DLK1, belongs to the EGF-like family of membrane proteins, which includes NOTCH receptors and their DSL-ligands. The molecular mechanisms by which DLK proteins regulate cell differentiation and proliferation processes are not fully established yet. In previous reports, we demonstrated that DLK1 interacts with itself and with specific EGF-like repeats of the NOTCH1 extracellular region involved in the binding to NOTCH1 canonical ligands. Moreover, the interaction of DLK1 with NOTCH1 caused an inhibition of basal NOTCH signaling in preadipocytes and mesenchymal multipotent cells. In this work, we demonstrate, for the first time, that DLK2 interacts with itself, with DLK1, and with the same NOTCH1 receptor region as DLK1 does. We demonstrate also that the interaction of DLK2 with NOTCH1 similarly results in an inhibition of NOTCH signaling in preadipocytes and Mouse Embryo fibloblasts. In addition, we demonstrate that a membrane DLK1 variant, lacking the sequence recognized by the protease TACE, also inhibits NOTCH signaling. Furthermore, both DLK1 and DLK2 are able to decrease NOTCH activity also when triggered by specific NOTCH ligands. However, the decrease in NOTCH signaling induced by overexpression of Dlk2 is reversed by the overexpression of Dlk1, and viceversa. We conclude that DLK1 and DLK2 act as inhibitory non-canonical protein ligands for the NOTCH1 receptor that modulate NOTCH signaling., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Macrophages present different Notch receptors and ligands on their surface. Following macrophage activation by LPS or other TLR ligands, Notch1 expression is upregulated. We report here that Notch signaling increases both basal and LPS-induced NF-kappaB activation, favoring the expression of genes implicated in the inflammatory response, such as the cytokines TNF-alpha and IL-6, or enzymes, such as iNOS. Delta4 seems to be the most effective ligand to induce Notch activation and increasing NF-kappaB transcriptional activity in macrophages. We show that Notch1 signaling promotes NF-kappaB translocation to the nucleus and DNA binding by increasing both phosphorylation of the IkappaB kinase alpha/beta complex and the expression of some NF-kappaB family members. Treatment of macrophages with the gamma-secretase inhibitor DAPT, which prevents the cleavage and activation of Notch receptors, inhibits all these processes, diminishing NF-kappaB activity following LPS stimulation. Additionally, we show that the active intracellular Notch fragment can directly interact with TNF-alpha and iNOS promoters. Our results suggest that Notch signaling results in an amplification of the macrophage-dependent inflammatory response by enhancing NF-kappaB signaling.

The Dlk1 gene appears to function as a regulator of adipogenesis. Adult Dlk1-deficient mice are obese, but adipose tissue still develops in transgenic mice overexpressing an Fc-dlk1 fusion protein, and neither type of genetically modified mice displays serious abnormalities. It was therefore possible that one yet unidentified gene might either compensate or antagonize for the absence or for overexpression, respectively, of Dlk1 in those animals. In database searches, we found a novel gene, EGFL9, encoding for a protein whose structural features are virtually identical to those of dlk1, suggesting it may function in a similar way. As dlk1 does, the protein encoded by EGFL9/Dlk2 affects adipogenesis of 3T3-L1 preadipocytes and mesenchymal C3H10T1/2 cells; however, it does so in an opposite way to that of dlk1. In addition, expression levels of both genes appear to be inversely correlated in both cell lines. Moreover, enforced changes in the expression of one gene affect the expression levels of the other. Our data suggest that adipogenesis may be modulated by the coordinated expression of Dlk1 and EGFL9/Dlk2.

Notch signaling has been extensively implicated in cell-fate determination along the development of the immune system. However, a role for Notch signaling in fully differentiated immune cells has not been clearly defined. We have analyzed the expression of Notch protein family members during macrophage activation. Resting macrophages express Notch-1, -2, and -4, as well as the Notch ligands Jagged-1 and -2. After treatment with LPS and/or IFN-gamma, we observed a p38 MAPK-dependent increase in Notch-1 and Jagged-1 mRNA and protein levels. To study the role of Notch signaling in macrophage activation, we forced the transient expression of truncated, active intracellular Notch-1 (Notch-IC) proteins in Raw 264.7 cells and analyzed their effects on the activity of transcription factors involved in macrophage activation. Notch-IC increased STAT-1-dependent transcription. Furthermore, Raw 264.7 Notch-IC stable transfectants increased STAT1-dependent transcription in response to IFN-gamma, leading to higher expression of IFN regulatory factor-1, suppressor of cytokine signaling-1, ICAM-1, and MHC class II proteins. This effect was independent from an increase of STAT1 Tyr or Ser phosphorylation. However, inducible NO synthase expression and NO production decreased under the same conditions. Our results show that Notch up-regulation and subsequent signaling following macrophage activation modulate gene expression patterns known to affect the function of mature macrophages.

The protein dlk, encoded by the Dlk1 gene, belongs to the Notch epidermal growth factor (EGF)-like family of receptors and ligands, which participate in cell fate decisions during development. The molecular mechanisms by which dlk regulates cell differentiation remain unknown. By using the yeast two-hybrid system, we found that dlk interacts with Notch1 in a specific manner. Moreover, by using luciferase as a reporter gene under the control of a CSL/RBP-Jk/CBF-1-dependent promoter in the dlk-negative, Notch1-positive Balb/c 14 cell line, we found that addition of synthetic dlk EGF-like peptides to the culture medium or forced expression of dlk decreases endogenous Notch activity. Furthermore, the expression of the gene Hes-1, a target for Notch1 activation, diminishes in confluent Balb/c14 cells transfected with an expression construct encoding for the extracellular EGF-like region of dlk. The expression of Dlk1 and Notch1 increases in 3T3-L1 cells maintained in a confluent state for several days, which is associated with a concomitant decrease in Hes-1 expression. On the other hand, the decrease of Dlk1 expression in 3T3-L1 cells by antisense cDNA transfection is associated with an increase in Hes-1 expression. These results suggest that dlk functionally interacts in vivo with Notch1, which may lead to the regulation of differentiation processes modulated by Notch1 activation and signaling, including adipogenesis.

The EGF-like membrane protein dlk plays a crucial role in the control of cell differentiation. Overexpression of the protein prevents, whereas inhibition of its expression increases, adipocyte differentiation of 3T3-L1 cells in response to Insulin-like Growth Factor I (IGF-1) or insulin. We have investigated whether dlk modulates the signaling pathways known to control this process. We found that the levels of dlk expression modulated signaling through the IGF-1 receptor, causing changes in the activation levels and kinetics of Extracellular-Regulated Kinase/Mitogen-Activated Protein Kinase (ERK/MAPK) that correlated with differentiation outcome. These changes occurred in response to IGF-1 or insulin but not in response to Epidermal Growth Factor. However, the levels of expression of IGF-1 receptor, or the activation of Insulin Receptor Substrate-1 in response to IGF-1, were not affected by the levels of dlk expression. Therefore, dlk appears to modulate ERK/MAPK signaling in response to specific differentiation signals., (Copyright 2002 Elsevier Science (USA).)

Levels of dlk, an EGF-like homeotic protein, are critical for several differentiation processes. Because growth and differentiation are, in general, exclusive of each other, and increasing evidence indicates that Dlk1 expression changes in tumorigenic processes, we studied whether dlk could also affect cell growth. We found that, in response to glucocorticoids, Balb/c 3T3 cells with diminished levels of dlk expression develop foci-like cells that have lost contact inhibition, display altered morphology, and grow faster than control cell lines. Balb/c 3T3 cells spontaneously growing more rapidly are also dlk-negative cells. Moreover, screening by the yeast two-hybrid system, using Dlk1 constructs as baits, resulted in the isolation of GAS1 and acrogranin cDNAs. Interestingly, these proteins are cysteine-rich molecules involved in the control of cell growth. Taken together, these observations suggest that dlk may participate in a network of interactions controlling how the cells respond to growth or differentiation signals.

The level of expression of dlk, an EGF-like protein possessing six EGF-like repeats in its extracellular region, is critical for 3T3-L1 fibroblasts to differentiate into adipocytes in response to IGF1. The mechanism of action of dlk is not well understood, but its localization on the cell membrane suggests that dlk may function as a receptor, as a ligand or as a regulatory protein modulating the binding, the signaling, or the expression of other molecules involved in cell differentiation and growth. In this work, we demonstrate, by using the Yeast Two-Hybrid system, that dlk interacts with itself through specific regions of its extracellular domain. The strongest interactions were observed between specific EGF-like repeats and between a non EFG-like region where unknown proteases act to generate soluble forms of dlk. These observations suggest that the interaction between two membrane dlk molecules belonging to the same or to different cells, or the interaction between soluble and membrane dlk variants, may be important to regulate dlk function.

A phytoene dehydrogenase-deficient mutant of Mucor circinelloides accumulating only phytoene was transformed with the gene encoding the corresponding enzyme (carB gene) of Phycomyces blakesleeanus. Carotenoids derived from phytoene were detected in the transformants showing that the P. blakesleeanus carB gene complements the M. circinelloides carB mutation. These newly formed carotenoids accumulated in low quantities, indicating that functional complementation was poor. carB mRNA molecules correctly transcribed were detected in the transformants, but they represented a small proportion of the total population of carB-derived mRNAs, mostly constituted by truncated transcripts and by transcripts longer than the transcript that is functional in Phycomyces. These results showed that the P. blakesleeanus carB gene was expressed in M. circinelloides and suggested that the poor complementation observed was owing, at least in part, to the lack of specificity in the recognition of the transcription initiation and termination signals of the P. blakesleeanus carB gene by the M. circinelloides transcriptional machinery.

Notch proteins participate in interactions between several cell types involved on the specification of numerous cell fates during development. We previously showed that enforced downregulation of Notch-1 expression prevented adipogenesis of 3T3-L1 cells. Since adipogenesis of 3T3-L1 cells can be induced by oncogenic ras, we studied whether this was also the case in 3T3-L1 cells with decreased levels of Notch-1 expression. We found that oncogenic ras induces transformation and not differentiation of 3T3-L1 cells with diminished levels of Notch-1. This result suggests that Notch-1 is implicated in the interpretation of signals leading to activation of p21 Ras.

Previous studies demonstrate that the delta-like (dlk) and preadipocyte factor 1 (Pref-1) genes encode similar proteins. Pref-1 is downregulated during adipocyte differentiation, and expression of ectopic Pref-1 inhibits adipogenesis. We explored whether dlk functions similarly to Pref-1 and studied the role of alternately spliced dlk variants encoding membrane-associated or -secreted forms. We also studied whether enforced downregulation of dlk/Pref-1 may enhance the differentiation response of non-committed cells. Ectopic expression of a potentially secreted dlk variant, conditioned media from dlk expressing cells or several individual epidermal-growth-factor-dlk peptides inhibited 3T3-L1 differentiation. This demonstrates that dlk and Pref-1 are functionally equivalent. dlk gene mRNA encoding for secreted variants decreased much faster than total dlk gene mRNA during differentiation of 3T3-L1 cells. In fact, total dlk or membrane-associated dlk protein expression increased during the first hours of differentiation. Cells sorted for lowest levels of dlk protein diminished or lost their ability to differentiate. These data suggest that membrane and secreted dlk protein variants play opposite roles in the control of adipogenesis. In addition, enforced downregulation of dlk protein expression in the weakly adipogenic Balb/c 3T3 cell line dramatically enhanced adipogenesis in response to insulin. These results indicate that dlk protein not only participates in processes leading to inhibition of adipogenesis but that the control of its expression and different spliced variants is essential for the adipogenic response to extracellular signals.

A close relationship exists between adipocyte differentiation of stromal cells and their capacity to support hematopoiesis. The molecular basis for this is unknown. We have studied whether dlk, an epidermal growth factor-like molecule that intervenes in adipogenesis and fetal liver hematopoiesis, affects both stromal cell adipogenesis and B-cell lymphopoiesis in an established pre-B-cell culture system. Pre-B-cell cultures require both soluble interleukin-7 (IL-7) and interactions with stromal cells to promote cell growth and prevent B-cell maturation or apoptosis. We found that BALB/c 3T3 fibroblasts express dlk and function as stromal cells. Transfection of these cells with antisense dlk decreased dlk expression and increased insulin-induced adipocytic differentiation. When antisense transfectants were used as stroma, IL-7 was no longer required to support the growth of pre-B cells and prevent maturation or apoptosis. Antisense dlk transfectants of S10 stromal cells also promoted pre-B-cell growth in the absence of IL-7. These results show that modulation of dlk on stromal cells can influence their adipogenesis and the IL-7 requirements of the pre-B cells growing in contact with them. These results indicate that dlk influences differentiation signals directed both to the stromal cells and to the lymphocyte precursors, suggesting that dlk may play an important role in the bone marrow hematopoietic environment.

Notch, a transmembrane receptor member of the homeotic epidermal growth factor-like family of proteins, participates in cell-to-cell signaling to control cell fate during development. Activated Notch-1 constructs lacking the extracellular region prevent differentiation of several mammalian cells in vitro. This effect, however, bypasses the normal mechanisms of cell-to-cell interactions in which Notch-1 participates. We investigated the role of Notch-1 in the hormone-induced adipocyte differentiation of 3T3-L1 fibroblasts, a paradigmatic model of adipogenesis that requires cell-to-cell contact. Unlike other differentiation models, Notch-1 expression and function were necessary conditions for adipogenesis. Impaired Notch-1 expression by antisense Notch-1 constructs prevented adipocyte differentiation. Strategies aimed at blocking putative Notch/ligand interactions also blocked adipogenesis, implicating Notch as a critical molecule in cell-to-cell signaling necessary for differentiation. Inhibition of Notch-1 expression or function decreased the expression of peroxisomal proliferator-activated receptors delta and gamma, transcription factors that control adipocyte differentiation and that are up-regulated at cell confluence. These results implicate Notch in the commitment of 3T3-L1 cells to undergo adipogenesis by controlling the expression of the principal regulators of this process.

Mutants of the filamentous fungus Mucor circinelloides altered in the synthesis of beta-carotene (genotype car) have been isolated by direct inspection of the color shown by the colonies derived from mutagenized spores. The mutants were analyzed for the carotenoid content in darkness and light and studied with respect to complementation in heterokaryons made by spheroplast fusion. The results revealed the existence of at least four complementation groups. The carB mutants are white and accumulate phytoene. The carR mutants are red and accumulate lycopene. The carP mutants are most likely disturbed in the synthesis of phytoene: two of them are white and do not accumulate carotenoids; another carP mutant is yellowish, probably because it is leaky. There are three yellowish mutants which belong to one or more complementation groups different from carB, carR, and carP. Two white mutants obtained in a single mutagenization step failed to complement with the carR and the carP mutants. The wild-type and the carB and carR mutants tested for M. circinelloides showed similar photoinduction., (Copyright 1997 Academic Press.)

By using a polymerase chain reaction based cloning strategy we isolated the gene (carB) encoding the enzyme phytoene dehydrogenase from Phycomyces blakesleeanus. The deduced protein, a 583 residue polypeptide, showed great similarity to carotenoid dehydrogenases from other fungi and bacteria, especially in the amino-terminal region. The main conserved regions found in other phytoene dehydrogenases, which are thought to be essential for the enzymatic activity, are present in the sequence from Phycomyces. Heterologous expression of the Phycomyces gene in Escherichia coli showed that, as in other fungi and bacteria, a single polypeptide catalyzes the four dehydrogenations that convert phytoene to lycopene. RNA measurements indicated that the level of expression of the phytoene dehydrogenase gene in wild-type mycelia increased in response to blue light. The kinetics of this increase in transcription of the gene after blue light induction (0.1 and 0.4 W/m2) exhibit a two-step (biphasic) dependence on fluence rate, suggesting that there could be two separate components involved in the reception of the low and high blue light signal. The presence of vitamin A in the medium stimulated transcript accumulation in the wild type and in some carotenogenic mutant strains. Diphenylamine, a phytoene dehydrogenase inhibitor, did not affect the level of transcription of this gene.

Carotenoid biosynthesis in wild type Mucor circinelloides has been investigated and the biochemical characterisation of the MS1 and MS9 mutant strains, impaired in carotenoid formation, carried out. In liquid cultures, all strains produced carotenoids (mainly beta-carotene, but also xi-carotene, lycopene and gamma-carotene) at the onset of stationary phase of growth. Carotenogenesis was light dependent. In liquid cultures carotenoid formation in wild type was affected by diphenylamine, which prevented desaturation, nicotine, resulting in reduced carotenoid levels, but CPTA caused an increase in the total carotenoid content but a reduced beta-carotene level, with the accumulation of lycopene and gamma-carotene. The mutant strains MS1 and MS9 contained only 5.0 and 11.5% of wild type carotenoid levels, respectively. Cell extracts of light-grown mycelia, incubated with 3(R)-[2-14C] mevalonic acid, produced beta-carotene, but incorporations into carotenoids were substantially reduced in the cell extracts of MS1 and MS9. Analysis of prenyl diphosphate intermediates indicated that, compared to wild type, geranylgeranyl diphosphate accumulated in MS1. MS9 extracts produced a larger amount of prenyl phosphates and a more even distribution of radioactivity from mevalonic acid into farnesyl and geranylgeranyl diphosphates. Squalene and long chain prenyl phosphates were formed by the cell extracts of all strains. It is proposed that the MS1 strain possesses a mutation in a gene responsible for phytoene formation, whilst a regulatory mutation, affecting prenyl transferase activities has occurred in MS9.