Targeting RET to induce medullary thyroid cancer cell apoptosis: an antagonistic interplay between PI3K/Akt and p38MAPK/caspase-8 pathways

Mutations in REarranged during Transfection (RET) receptor tyrosine, followed by the oncogenic activation of RET kinase is responsible for the development of medullary thyroid carcinoma (MTC) that responds poorly to conventional chemotherapy. Targeting RET, therefore, might be useful in tailoring surveillance of MTC patients. Here we showed that theaflavins, the bioactive components of black tea, successfully induced apoptosis in human MTC cell line, TT, by inversely modulating two molecular pathways: (i) stalling PI3K/Akt/Bad pathway that resulted in mitochondrial transmembrane potential (MTP) loss, cytochrome-c release and activation of the executioner caspases-9 and -3, and (ii) upholding p38MAPK/caspase-8/caspase-3 pathway via inhibition of Ras/Raf/ERK. Over-expression of either constitutively active myristoylated-Akt-cDNA (Myr-Akt-cDNA) or dominant-negative-caspase-8-cDNA (Dn-caspase-8-cDNA) partially blocked theaflavin-induced apoptosis, while co-transfection of Myr-Akt-cDNA and Dn-caspase-8-cDNA completely eradicated the effect of theaflavins thereby negating the possibility of existence of other pathways. A search for the upstream signaling revealed that theaflavin-induced disruption of lipid raft caused interference in anchorage of RET in lipid raft that in turn stalled phosphorylation of Ras and PI3Kinase. In such anti-survival cellular micro-environment, pro-apoptotic signals were triggered to culminate into programmed death of MTC cell. These findings not only unveil a hitherto unexplained mechanism underlying theaflavin-induced MTC death, but also validate RET as a promising and potential target for MTC therapy.     

Investigations on Modulating Effect of Vanadium Supplementation on Growth and Metabolism Through Improved Immune Response,Antioxidative Profile and Endocrine Variables in Hariana heifers

Biological Trace Element Research - This study was conducted to investigate the effect of vanadium (V) supplementation on growth, metabolism, antioxidant, and immunological and endocrine variables...     

Frizzled-4 is required for normal bone acquisition despite compensation by Frizzled-8

The activation of the Wnt/β-catenin signaling pathway is critical for skeletal development but surprisingly little is known about the requirements for the specific frizzled (Fzd) receptors that recognize Wnt ligands. To define the contributions of individual Fzd proteins to osteoblast function, we profiled the expression of all 10 mammalian receptors during calvarial osteoblast differentiation. Expression of Fzd4 was highly upregulated during in vitro differentiation and therefore targeted for further study. Mice lacking Fzd4 in mature osteoblasts had normal cortical bone structure but reduced cortical tissue mineral density and also exhibited an impairment in the femoral trabecular bone acquisition that was secondary to a defect in the mineralization process. Consistent with this observation, matrix mineralization, markers of osteoblastic differentiation, and the ability of Wnt3a to stimulate the accumulation of β-catenin were reduced in cultures of calvarial osteoblasts deficient for Fzd4. Interestingly, Fzd4-deficient osteoblasts exhibited an increase in the expression of Fzd8 both in vitro and in vivo, which suggests that the two receptors may exhibit overlapping functions. Indeed, ablating a single Fzd8 allele in osteoblast-specific Fzd4 mutants produced a more severe effect on bone acquisition. Taken together, our data indicate that Fzd4 is required for normal bone development and mineralization despite compensation from Fzd8.     

185 Vascular endothelial growth factor receptor-2 (VEGFR2): structure and functional relationship

Vascular endothelial growth factor (VEGF), is expressed in the vicinity of sprouting vessels and its receptor (VEGF-R2/Flk-1/kdr) on the angioblasts and new vessels, and both are required for vasculogenesis and angiogenesis. VEGFR2, also called as KDR or Flk-1, is identified as an early marker for endothelial cell progenitors, whose expression is restricted to endothelial cells in vivo. VEGFR2 consists of extracellular (7-Ig-like sub-domains), transmembrane and cytoplasmic domains. In order to understand the structure–functional relationship and signal transduction process of VEGFR2, we have examined their amino acid sequences from a wide range of species including mammals, birds, Zebrafish and also computed the phylogenetic tree, secondary and domain structures. Phylogeny constructed using Maximum Parsimony tree software MEGA-5 version suggested an interesting sequence similarity between Zebrafish and Gallus, closeness between human, rat, horse and pig. Strong homology in amino acids sequences was observed between the species, such as human, Macaca mulatta, gorilla, etc, and small variations in Zebrafish and zebrafinch. The Arg and Asp residues which are involved in forming salt bridges are evolutionarily conserved from Zebrafish to human in D7 domain of VEGFR2, indicating their functional importance in VEGFR activity. Amino acids, tyrosine in the extracellular loops and cysteines involved in disulphide bridges of VEGFR2, are highly conserved suggesting their importance during ligand binding, the details of which will be discussed.     

Putative osmosensor – OsHK3b – a histidine kinase protein from rice shows high structural conservation with its ortholog AtHK1 from Arabidopsis

Prokaryotes and eukaryotes respond to various environmental stimuli using the two-component system (TCS). Essentially, it consists of membrane-bound histidine kinase (HK) which senses the stimuli and further transfers the signal to the response regulator, which in turn, regulates expression of various target genes. Recently, sequence-based genome wide analysis has been carried out in Arabidopsis and rice to identify all the putative members of TCS family. One of the members of this family i.e. AtHK1, (a putative osmosensor, hybrid-type sensory histidine kinase) is known to interact with AtHPt1 (phosphotransfer proteins) in Arabidopsis. Based on predicted rice interactome network (PRIN), the ortholog of AtHK1 in rice, OsHK3b, was found to be interacting with OsHPt2. The analysis of amino acid sequence of AtHK1 showed the presence of transmitter domain (TD) and receiver domain (RD), while OsHK3b showed presence of three conserved domains namely CHASE (signaling domain), TD, and RD. In order to elaborate on structural details of functional domains of hybrid-type HK and phosphotransfer proteins in both these genera, we have modeled them using homology modeling approach. The structural motifs present in various functional domains of the orthologous proteins were found to be highly conserved. Binding analysis of the RD domain of these sensory proteins in Arabidopsis and rice revealed the role of various residues such as histidine in HPt protein which are essential for their interaction.     

Oxidized Low Density Lipoprotein Induced Caspase-1 Mediated Pyroptotic Cell Death in Macrophages: Implication in Lesion Instability?

Background

Macrophage death in advanced lesion has been confirmed to play an important role in plaque instability. However, the mechanism underlying lesion macrophage death still remains largely unknown.

Methods and Results

Immunohistochemistry showed that caspase-1 activated in advanced lesion and co-located with macrophages and TUNEL positive reaction. In in-vitro experiments showed that ox-LDL induced caspase-1 activation and this activation was required for ox-LDL induced macrophages lysis, IL-1β and IL-18 production as well as DNA fragmentation. Mechanism experiments showed that CD36 and NLRP3/caspase-1/pathway involved in ox-LDL induced macrophage pyroptosis.

Conclusion

Our study here identified a novel cell death, pyroptosis in ox-LDL induced human macrophage, which may be implicated in lesion macrophages death and play an important role in lesion instability.