Differentiation dependent expression of TRPA1 and TRPM8 channels in IMR‐32 human neuroblastoma cells

TRPA1 and TRPM8 are transient receptor potential (TRP) channels involved in sensory perception. TRPA1 is a non‐selective calcium permeable channel activated by irritants and proalgesic agents. TRPM8 reacts to chemical cooling agents such as menthol. The human neuroblastoma cell line IMR‐32 undergoes a remarkable differentiation in response to treatment with 5‐bromo‐2‐deoxyuridine. The cells acquire a neuronal morphology with increased expression of N‐type voltage gated calcium channels and neurotransmitters. Here we show using RT‐PCR, that mRNA for TRPA1 and TRPM8 are strongly upregulated in differentiating IMR‐32 cells. Using whole cell patch clamp recordings, we demonstrate that activators of these channels, wasabi, allyl‐isothiocyanate (AITC) and menthol activate membrane currents in differentiated cells. Calcium imaging experiments demonstrated that AITC mediated elevation of intracellular calcium levels were attenuated by ruthenium red, spermine, and HC‐030031 as well as by siRNA directed against the channel. This indicates that the detected mRNA level correlate with the presence of functional channels of both types in the membrane of differentiated cells. Although the differentiated IMR‐32 cells responded to cooling many of the cells showing this response did not respond to TRPA1/TRPM8 channel activators (60% and 90% for AITC and menthol respectively). Conversely many of the cells responding to these activators did not respond to cooling (30%). This suggests that these channels have also other functions than cold perception in these cells. Furthermore, our results suggest that IMR‐32 cells have sensory characteristics and can be used to study native TRPA1 and TRPM8 channel function as well as developmental expression. J. Cell. Physiol. 221: 67–74, 2009. © 2009 Wiley‐Liss, Inc     

Progressive effects of N‐myc deficiency on proliferation,neurogenesis, and morphogenesis in the olfactory epithelium

N‐myc belongs to the myc proto‐oncogene family, which is involved in numerous cellular processes such as proliferation, growth, apoptosis, and differentiation. Conditional deletion of N‐myc in the mouse nervous system disrupted brain development, indicating that N‐myc plays an essential role during neural development. How the development of the olfactory epithelium and neurogenesis within are affected by the loss of N‐myc has, however, not been determined. To address these issues, we examined an N‐myc^Foxg1Cre conditional mouse line, in which N‐myc is depleted in the olfactory epithelium. First changes in N‐myc mutants were detected at E11.5, with reduced proliferation and neurogenesis in a slightly smaller olfactory epithelium. The phenotype was more pronounced at E13.5, with a complete lack of Hes5‐positive progenitor cells, decreased proliferation, and neurogenesis. In addition, stereological analyses revealed reduced cell size of post‐mitotic neurons in the olfactory epithelium, which contributed to a smaller olfactory pit. Furthermore, we observed diminished proliferation and neurogenesis also in the vomeronasal organ, which likewise was reduced in size. In addition, the generation of gonadotropin‐releasing hormone neurons was severely reduced in N‐myc mutants. Thus, diminished neurogenesis and proliferation in combination with smaller neurons might explain the morphological defects in the N‐myc depleted olfactory structures. Moreover, our results suggest an important role for N‐myc in regulating ongoing neurogenesis, in part by maintaining the Hes5‐positive progenitor pool. In summary, our results provide evidence that N‐myc deficiency in the olfactory epithelium progressively diminishes proliferation and neurogenesis with negative consequences at structural and cellular levels. © 2013 The Authors. Developmental Neurobiology Published by Wiley Periodicals, Inc. Develop Neurobiol 74: 643–656, 2014     

Inhibition of the CSF‐1 receptor sensitizes ovarian cancer cells to cisplatin

Ovarian cancer is one of the most common female malignancies, and cisplatin‐based chemotherapy is routinely used in locally advanced ovarian cancer patients. Acquired or de novo cisplatin resistance remains the barrier to patient survival, and the mechanisms of cisplatin resistance are still not well understood. In the current study, we found that colony‐stimulating‐factor‐1 receptor (CSF‐1R) was upregulated in cisplatin‐resistant SK‐OV‐3 and CaoV‐3 cells. Colony‐stimulating‐factor‐1 receptor knockdown suppressed proliferation and enhanced apoptosis in cisplatin‐resistant SK‐OV‐3 and CaoV‐3 cells. However, CSF‐1R overexpression had inverse effects. While parental SK‐OV‐3 and CaoV‐3 cells were more resistant to cisplatin after CSF‐1R overexpression, CSF‐1R knockdown in SK‐OV‐3 and CaoV‐3 cells promoted cisplatin sensitivity. Overexpression and knockdown studies also showed that CSF‐1R significantly promoted active AKT and ERK1/2 signalling pathways in cisplatin‐resistant cells. Furthermore, a combination of cisplatin and CSF‐1R inhibitor effectively inhibited tumour growth in xenografts. Taken together, our results provide the first evidence that CSF‐1R inhibition can sensitize cisplatin‐refractory ovarian cancer cells. This study may help to increase understanding of the molecular mechanisms underlying cisplatin resistance in tumours.     

Bone marrow‐derived mesenchymal stem cells mitigate caspase‐3 and 8‐hydroxy proline induced via β‐adrenergic agonist in pulmonary injured rats

Estimating the ability of bone marrow‐derived mesenchymal stem cells (BM‐MSCs) to alleviate pulmonary injury induced via isoproterenol (ISP). ISP was injected in a dose of (100 mg/kg, subcutaneously twice at an interval of 24 h). One month post BM‐MSCs transplantation by intravenous injection, pulmonary oxidative stress was assessed, and Western blot analyses and histopathological investigations were conducted. Compared with the normal control group, BM‐MSCs transplantation significantly decreased the expression of pulmonary anti‐oxidative stress marker. Western blot analysis revealed that ISP significantly reduced the protein expression of the anti‐oxidative stress marker nuclear related factor‐2 (Nrf2). However, the apoptotic marker (caspase‐3) and collagen content marker (8‐hydroxyproline) were markedly elevated. These biochemical markers were confirmed by histopathological investigations. Finally, it was demonstrated that BM‐MSCs transplantation showed a superior effect in improving pulmonary function through alleviating oxidative stress, apoptosis, and collagen content.     

Protein kinase Cδ and caspase‐3 modulate TRAIL‐induced apoptosis in breast tumor cells

This report describes that protein kinase C delta (PKCδ) overexpression prevents TRAIL‐induced apoptosis in breast tumor cells; however, the regulatory mechanism(s) involved in this phenomenon is(are) incompletely understood. In this study, we have shown that TRAIL‐induced apoptosis was significantly inhibited in PKCδ overexpressing MCF‐7 (MCF7/PKCδ) cells. Our data reveal that PKCδ inhibits caspase‐8 activation, a first step in TRAIL‐induced apoptosis, thus preventing TRAIL‐induced apoptosis. Inhibition of PKCδ using rottlerin or PKCδ siRNA reverses the inhibitory effect of PKCδ on caspase‐8 activation leading to TRAIL‐induced apoptosis. To determine if caspase‐3‐induced PKCδ cleavage reverses its inhibition on caspase‐8, we developed stable cell lines that either expresses wild‐type PKCδ (MCF‐7/cas‐3/PKCδ) or caspase‐3 cleavage‐resistant PKCδ mutant (MCF‐7/cas‐3/PKCδ mut) utilizing MCF‐7 cells expressing caspase‐3. Cells that overexpress caspase‐3 cleavage‐resistant PKCδ mutant (MCF‐7/cas‐3/PKCδmut) significantly inhibited TRAIL‐induced apoptosis when compared to wild‐type PKCδ (MCF‐7/cas‐3/PKCδ) expressing cells. In MCF‐7/cas‐3/PKCδmut cells, TRAIL‐induced caspase‐8 activation was blocked leading to inhibition of apoptosis when compared to wild‐type PKCδ (MCF‐7/cas‐3/PKCδ) expressing cells. Together, these results strongly suggest that overexpression of PKCδ inhibits caspase‐8 activation leading to inhibition of TRAIL‐induced apoptosis and its inhibition by rottlerin, siRNA, or cleavage by caspase‐3 sensitizes cells to TRAIL‐induced apoptosis. Clinically, PKCδ overexpressing tumors can be treated with a combination of PKCδ inhibitor(s) and TRAIL as a new treatment strategy. J. Cell. Biochem. 111: 979–987, 2010. © 2010 Wiley‐Liss, Inc.     

Mitochondrial apoptosis is dispensable for NLRP3 inflammasome activation but non‐apoptotic caspase‐8 is required for inflammasome priming

A current paradigm proposes that mitochondrial damage is a critical determinant of NLRP3 inflammasome activation. Here, we genetically assess whether mitochondrial signalling represents a unified mechanism to explain how NLRP3 is activated by divergent stimuli. Neither co‐deletion of the essential executioners of mitochondrial apoptosis BAK and BAX, nor removal of the mitochondrial permeability transition pore component cyclophilin D, nor loss of the mitophagy regulator Parkin, nor deficiency in MAVS affects NLRP3 inflammasome function. In contrast, caspase‐8, a caspase essential for death‐receptor‐mediated apoptosis, is required for efficient Toll‐like‐receptor‐induced inflammasome priming and cytokine production. Collectively, these results demonstrate that mitochondrial apoptosis is not required for NLRP3 activation, and highlight an important non‐apoptotic role for caspase‐8 in regulating inflammasome activation and pro‐inflammatory cytokine levels.     

Replication of IMR‐32‐adapted JC virus clones in human embryonic kidney cells

It has been difficult to study JCV replication because of its restricted host range. In this study, JCV replication was examined using different clones in 293 cells. RT‐PCR assay revealed that large T antigen expression in cells transfected with IMR‐32‐adapted JCVs was significantly greater than in those transfected with Mad‐1 or CY. DNA replication assay and viral load verified that the IMR‐32‐adapted JCVs were replication‐competent in 293 cells, but not Mad‐1 or CY JCVs. These results suggest that a 293 culture system with IMR‐32‐adapted JCVs may be a useful tool for assessing replication of JCV in vitro.     

Differential expression of nuclear matrix proteins during the differentiation of human neuroblastoma SK‐N‐SH cells induced by retinoic acid

To investigate the alteration of nuclear matrix proteins (NMPs) during the differentiation of neuroblastoma SK‐N‐SH cells induced by retinoic acid (RA), differentiation markers were detected by immunocytochemistry and NMPs were selectively extracted and subjected to two‐dimensional gel electrophoresis analysis. Immunocytochemical observation demonstrated that the expression of neuronal markers was up‐regulated in SK‐N‐SH cells following RA treatment. Meanwhile, 52 NMPs (41 of which were identified) changed significantly during SK‐N‐SH differentiation; four of these NMPs were further confirmed by immunoblotting. This study suggests that the differentiation of neuroblastoma cells was accompanied by the altered expression of neuronal markers and NMPs. The presence of some differentially expressed NMPs was related to the proliferation and differentiation of neuroblastomas. Our results may help to reveal the relationship between NMPs and neuroblastoma carcinogenesis and reversion, as well as elucidate the regulatory principals driving neural cell proliferation and differentiation. J. Cell. Biochem. 106: 849–857, 2009. © 2009 Wiley‐Liss, Inc.     

Inhibition of CaMKII-mediated c-FLIP expression sensitizes malignant melanoma cells to TRAIL-induced apoptosis

TRAIL can induce apoptosis in melanoma cells and thus may offer new hope for melanoma therapy. However, many melanoma cells are resistant to TRAIL. To examine molecular mechanisms in cell resistance, we analyzed TRAIL-induced DISC in TRAIL-sensitive melanoma cells and showed that apoptosis-initiating caspase-8 and caspase-10 were recruited to the DISC where they became activated through autocatalytical cleavage, leading to apoptosis through cleavage of downstream substrates such as caspase-3 and DFF45. In TRAIL-resistant melanoma cells, however, c-FLIP proteins were recruited to the DISC, resulting in the inhibition of caspase-8 and caspase-10 cleavage in the DISC. Both calmodulin-dependent protein kinase II (CaMKII) protein and enzymatic activity were upregulated in resistant cells and CaMKII inhibitor KN-93 downregulated expression of c-FLIP proteins, thus sensitizing resistant cells to TRAIL-induced apoptosis. Transfection of CaMKII cDNA in sensitive melanoma cells resulted in cell resistance to TRAIL, where transfection of CaMKII dominant-negative cDNA in resistant cells restored TRAIL sensitivity in cells. These results indicate that the CaMKII-mediated pathway for c-FLIP upregulation protects melanoma cells from TRAIL-induced apoptosis and targeting this pathway may provide novel therapeutic strategies in treatment of melanomas.     

Dynamic expression of N‐myc in mouse embryonic development using an enhanced green fluorescent protein reporter gene in the N‐myc locus

N‐myc belongs to the Myc oncogene family and plays an essential role in mammalian embryonic development. The expression of N‐myc is dynamically regulated during embryonic development; however, its expression pattern has not been well characterized due to the lack of a suitable animal model. In this paper, a genetically modified mouse model was generated in which the enhanced green fluorescent protein (EGFP) coding sequence was inserted into the N‐myc locus, so that endogenous N‐myc expression could be traced by the signal of EGFP. The EGFP signal in the transgenic mouse was confirmed to be consistent with the expression pattern of endogenous N‐myc by fluorescence microscopy and immunohistochemical staining. Furthermore, the spatial and temporal expression of EGFP was observed in the central and peripheral nervous system, heart, lung and kidney, given the known indispensable role of N‐myc in their formation. EGFP was also strongly detected in the liver, paranephros and the epithelium of the intestine. The EGFP signal can be used to trace N‐myc expression in this transgenic mouse model. N‐myc expression was observed in specific locations and cell lineages, and dynamically changed during embryonic development. The changing N‐myc expression pattern seen in mouse embryonic development and the animal model described in this paper provide important insights and a new tool to research N‐myc function.     

Insulin‐like growth factor‐I prevents caspase‐mediated apoptosis in Schwann cells

Both neurons and glia succumb to programmed cell death (PCD) when deprived of growth factors at critical periods in development or following injury. Insulin‐like growth factor‐I (IGF‐I) prevents apoptosis in neurons in vitro. To investigate whether IGF‐I can protect Schwann cells (SC) from apoptosis, SC were harvested from postnatal day 3 rats and maintained in serum‐containing media until confluency. When cells were switched to serum‐free defined media (DM) for 12–72 h, they underwent PCD. Addition of insulin or IGF‐I prevented apoptosis. Bisbenzamide staining revealed nuclear condensation and formation of apoptotic bodies in SC grown in DM alone, but SC grown in DM plus IGF‐I had normal nuclear morphology. The phosphatidylinositol 3‐kinase (PI 3‐K) inhibitor LY294002 blocked IGF‐I–mediated protection. Caspase‐3 activity was rapidly activated upon serum withdrawal in SC, and the caspase inhibitor BAF blocked apoptosis. These results suggest that IGF‐I rescues SC from apoptosis via PI 3‐K signaling which is upstream from caspase activation. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 540–548, 1999     

Tranilast attenuates methotrexate‐induced renal and hepatic toxicities: Role of apoptosis‐induced tissue proliferation

Drug‐induced organ toxicity is a frequently encountered obstacle in the field of medical practice that limits the use of numerous pharmacologically valuable drugs. Methotrexate (MTX)‐induced organ toxicity is unfortunately the rate‐limiting factor for its clinical application. In the current study, MTX injection induced significant renal and hepatic toxicities manifested on functional, biochemical, and histopathological scales. This was associated with a significant elevation in both renal and hepatic contents of TNF‐related apoptosis–inducing ligand (TRAIL) and caspase‐8, biomarkers of tissue apoptosis. Inline, immunohistochemical analysis confirmed that tissue increased expression of Ki67 as a biomarker of tissue regeneration in both organs. Tranilast (TRAN) is a small molecular weight anti‐inflammatory and antiallergic agent. TRAN's coadministration with MTX in the current study induced a significant tissue recovery via modulation of TRAIL/caspase‐8 signaling and modulation of apoptosis‐induced tissue proliferation confirmed by quantification of Ki67 expression. In conclusion, TRAN can be proposed as an effective drug to attenuate MTX‐induced organ toxicity via modulation of apoptosis‐induced tissue proliferation pathway.     

Purification and characterization of active caspase‐14 from human epidermis and development of the cleavage site‐directed antibody

Restricted expression of caspase‐14 in differentiating keratinocytes suggests the involvement of caspase‐14 in terminal differentiation. We purified active caspase‐14 from human cornified cells with sequential chromatographic procedures. Specific activity increased 764‐fold with a yield of 9.1%. Purified caspase‐14 revealed the highest activity on WEHD‐methylcoumaryl‐amide (MCA), although YVAD‐MCA, another caspase‐1 substrate, was poorly hydrolyzed. The purified protein was a heterodimer with 17 and 11 kDa subunits. N‐terminal and C‐terminal analyses demonstrated that the large subunit consisted of Ser⁶‐Asp¹⁴⁶ and N‐terminal of small subunit was identified as Lys¹⁵³. We successfully developed an antiserum (anti‐h14D146) directed against the Asp¹⁴⁶ cleavage site, which reacted only with active caspase‐14 but not with procaspase‐14. Furthermore we confirmed that anti‐h14D146 did not show any reactivity to the active forms of other caspases. Immunohistochemical analysis demonstrated that anti‐h14D146 staining was mostly restricted to the cornified layer and co‐localized with some of the TUNEL positive‐granular cells in the normal human epidermis. UV radiation study demonstrated that caspase‐3 was activated and co‐localized with TUNEL‐positive cells in the middle layer of human epidermis. In contrast, we could not detect caspase‐14 activation in response to UV. Our study revealed tightly regulated action of caspase‐14, in which only the terminal differentiation of keratinocytes controls its activation process. J. Cell. Biochem. 109: 487–497, 2010. © 2009 Wiley‐Liss, Inc.     

Solution NMR characterization of WT CXCL8 monomer and dimer binding to CXCR1 N‐terminal domain

Chemokine CXCL8 and its receptor CXCR1 are key mediators in combating infection and have also been implicated in the pathophysiology of various diseases including chronic obstructive pulmonary disease (COPD) and cancer. CXCL8 exists as monomers and dimers but monomer alone binds CXCR1 with high affinity. CXCL8 function involves binding two distinct CXCR1 sites – the N‐terminal domain (Site‐I) and the extracellular/transmembrane domain (Site‐II). Therefore, higher monomer affinity could be due to stronger binding at Site‐I or Site‐II or both. We have now characterized the binding of a human CXCR1 N‐terminal domain peptide (hCXCR1Ndp) to WT CXCL8 under conditions where it exists as both monomers and dimers. We show that the WT monomer binds the CXCR1 N‐domain with much higher affinity and that binding is coupled to dimer dissociation. We also characterized the binding of two CXCL8 monomer variants and a trapped dimer to two different hCXCR1Ndp constructs, and observe that the monomer binds with ～10‐ to 100‐fold higher affinity than the dimer. Our studies also show that the binding constants of monomer and dimer to the receptor peptides, and the dimer dissociation constant, can vary significantly as a function of pH and buffer, and so the ability to observe WT monomer peaks is critically dependent on NMR experimental conditions. We conclude that the monomer is the high affinity CXCR1 agonist, that Site‐I interactions play a dominant role in determining monomer vs. dimer affinity, and that the dimer plays an indirect role in regulating monomer function.