Identification of plants' functional counterpart of the metazoan mediator of DNA Damage checkpoint 1.

Induction of DNA damage triggers rapid phosphorylation of the histone H2A.X (γH2A.X). In animals, mediator of DNA damage checkpoint 1 (MDC1) binds γH2A.X through a tandem BRCA1 carboxyl-terminal (tBRCT) domain and mediates recruitment of downstream effectors of DNA damage response (DDR). However, readers of this modification in plants have remained elusive. We show that from the Arabidopsis BRCT domain proteome, BCP1-4 proteins with tBRCT domains are involved in DDR. Through its tBRCT domain BCP4 binds γH2A.X in vitro and localizes to DNA damage-induced foci in an H2A.X-dependent manner. BCP4 also contains a domain that interacts directly with NBS1 and thus acts as a functional counterpart of MDC1. We also show that BCP1, that contains two tBRCT domains, co-localizes with γH2A.X but it does not bind γH2A.X suggesting functional similarity with human PAXIP1. A phylogenetic analysis supports that PAXIP1 and MDC1 in metazoa and their plant counterparts evolved independently from common ancestors with tBRCT domains. Collectively, our study reveals missing components and provides mechanistic and evolutionary insights into plant DDR.

Extremophilic red algae as models for understanding adaptation to hostile environments and the evolution of eukaryotic life on the early earth.

Extremophiles have always garnered great interest because of their exotic lifestyles and ability to thrive at the physical limits of life. In hot springs environments, the Cyanidiophyceae red algae are the only photosynthetic eukaryotes able to live under extremely low pH (0-5) and relatively high temperature (35ºC to 63ºC). These extremophiles live as biofilms in the springs, inhabit acid soils near the hot springs, and form endolithic populations in the surrounding rocks. Cyanidiophyceae represent a remarkable source of knowledge about the evolution of extremophilic lifestyles and their genomes encode specialized enzymes that have applied uses. Here we review the evolutionary origin, taxonomy, genome biology, industrial applications, and use of Cyanidiophyceae as genetic models. Currently, Cyanidiophyceae comprise a single order (Cyanidiales), three families, four genera, and nine species, including the well-known Cyanidioschyzon merolae and Galdieria sulphuraria. These algae have small, gene-rich genomes that are analogous to those of prokaryotes they live and compete with. There are few spliceosomal introns and evidence exists for horizontal gene transfer as a driver of local adaptation to gain access to external fixed carbon and to extrude toxic metals. Cyanidiophyceae offer a variety of commercial opportunities such as phytoremediation to detoxify contaminated soils or waters and exploitation of their mixotrophic lifestyles to support the efficient production of bioproducts such as phycocyanin and floridosides. In terms of exobiology, Cyanidiophyceae are an ideal model system for understanding the evolutionary effects of foreign gene acquisition and the interactions between different organisms inhabiting the same harsh environment on the early Earth. Finally, we describe ongoing research with C. merolae genetics and summarize the unique insights they offer to the understanding of algal biology and evolution.

Revised classification of the Cyanidiophyceae based on plastid genome data with descriptions of the Cavernulicolales ord. nov. and Galdieriales ord. nov. (Rhodophyta).

The Cyanidiophyceae, an extremophilic red algal class, is distributed worldwide in extreme environments. Species grow either in acidic hot environments or in dim light conditions (e.g., "cave Cyanidium"). The taxonomy and classification systems are currently based on morphological, eco-physiological, and molecular phylogenetic characters; however, previous phylogenetic results showed hidden diversity of the Cyanidiophyceae and suggested a revision of the classification system. To clarify phylogenetic relationships within this red algal class, we employ a phylogenomic approach based on 15 plastomes (10 new) and 15 mitogenomes (seven new). Our phylogenies show consistent relationships among four lineages (Galdieria, "cave Cyanidium", Cyanidium, and Cyanidioschyzon lineages). Each lineage is distinguished by organellar genome characteristics. The "cave Cyanidium" lineage is a distinct clade that diverged after the Galdieria clade but within a larger monophyletic clade that included the Cyanidium and Cyanidioschyzon lineages. Because the "cave Cyanidium" lineage is a mesophilic lineage that differs substantially from the other three thermoacidophilic lineages, we describe it as a new order (Cavernulicolales). Based on this evidence, we reclassified the Cyanidiophyceae into four orders: Cyanidiales, Cyanidioschyzonales, Cavernulicolales ord. nov., and Galdieriales ord. nov. The genetic distance among these four orders is comparable to, or greater than, the distances found between other red algal orders and subclasses. Three new genera (Cavernulicola, Gronococcus, Sciadococcus), five new species (Galdieria javensis, Galdieria phlegrea, Galdieria yellowstonensis, Gronococcus sybilensis, Sciadococcus taiwanensis), and a new nomenclatural combination (Cavernulicola chilensis) are proposed.

Group II intron and repeat-rich red algal mitochondrial genomes demonstrate the dynamic recent history of autocatalytic RNAs.

BACKGROUND: Group II introns are mobile genetic elements that can insert at specific target sequences, however, their origins are often challenging to reconstruct because of rapid sequence decay following invasion and spread into different sites. To advance understanding of group II intron spread, we studied the intron-rich mitochondrial genome (mitogenome) in the unicellular red alga, Porphyridium. RESULTS: Analysis of mitogenomes in three closely related species in this genus revealed they were 3-6-fold larger in size (56-132 kbp) than in other red algae, that have genomes of size 21-43 kbp. This discrepancy is explained by two factors, group II intron invasion and expansion of repeated sequences in large intergenic regions. Phylogenetic analysis demonstrates that many mitogenome group II intron families are specific to Porphyridium, whereas others are closely related to sequences in fungi and in the red alga-derived plastids of stramenopiles. Network analysis of intron-encoded proteins (IEPs) shows a clear link between plastid and mitochondrial IEPs in distantly related species, with both groups associated with prokaryotic sequences. CONCLUSION: Our analysis of group II introns in Porphyridium mitogenomes demonstrates the dynamic nature of group II intron evolution, strongly supports the lateral movement of group II introns among diverse eukaryotes, and reveals their ability to proliferate, once integrated in mitochondrial DNA.

Amoeba Genome Reveals Dominant Host Contribution to Plastid Endosymbiosis.

Eukaryotic photosynthetic organelles, plastids, are the powerhouses of many aquatic and terrestrial ecosystems. The canonical plastid in algae and plants originated >1 Ga and therefore offers limited insights into the initial stages of organelle evolution. To address this issue, we focus here on the photosynthetic amoeba Paulinella micropora strain KR01 (hereafter, KR01) that underwent a more recent (∼124 Ma) primary endosymbiosis, resulting in a photosynthetic organelle termed the chromatophore. Analysis of genomic and transcriptomic data resulted in a high-quality draft assembly of size 707 Mb and 32,361 predicted gene models. A total of 291 chromatophore-targeted proteins were predicted in silico, 208 of which comprise the ancestral organelle proteome in photosynthetic Paulinella species with functions, among others, in nucleotide metabolism and oxidative stress response. Gene coexpression analysis identified networks containing known high light stress response genes as well as a variety of genes of unknown function ("dark" genes). We characterized diurnally rhythmic genes in this species and found that over 49% are dark. It was recently hypothesized that large double-stranded DNA viruses may have driven gene transfer to the nucleus in Paulinella and facilitated endosymbiosis. Our analyses do not support this idea, but rather suggest that these viruses in the KR01 and closely related P. micropora MYN1 genomes resulted from a more recent invasion.

STAT3 activation in microglia increases pericyte apoptosis in diabetic retinas through TNF-ɑ/AKT/p70S6 kinase signaling.

Diabetic retinopathy (DR) is one of the vascular complications associated with diabetes mellitus. Pericyte loss is an early characteristic phenomenon in DR. However, the mechanism by which pericyte apoptosis occurs in DR is not fully understood. We have focused on the increased STAT3 activation in diabetic retinas because STAT3 activation is associated with inflammation, and persistent chronic inflammation is closely related to retinal lesions. In this study, we demonstrated that STAT3 was activated by IFN-γ and IL-6 that highly expressed in diabetic retinas. We identified TNF-α as a potent inducer of pericyte apoptosis in diabetic retinas from the gene expression analysis and found that STAT3 activation in microglia increased TNF-α expression in the diabetic retinas. We also demonstrated that increased TNF-α expression in microglia caused pericyte apoptosis through downregulating AKT/p70S6 kinase signaling. Moreover, we took advantage of mice lacking STAT3 in microglia and demonstrated that STAT3 ablation in microglia reduced the pericyte apoptosis and TNF-α expression in the diabetic retinas. These results suggest that STAT3 activation in microglia plays an important role in pericyte apoptosis in the diabetic retinas through increased TNF-α expression and provide STAT3 activation in microglia as a potential therapeutic target for preventing pericyte loss in DR.

The acidic tumor microenvironment enhances PD-L1 expression via activation of STAT3 in MDA-MB-231 breast cancer cells.

Tumor acidosis, a common phenomenon in solid cancers such as breast cancer, is caused by the abnormal metabolism of cancer cells. The low pH affects cells surrounding the cancer, and tumor acidosis has been shown to inhibit the activity of immune cells. Despite many previous studies, the immune surveillance mechanisms are not fully understood. We found that the expression of PD-L1 was significantly increased under conditions of extracellular acidosis in MDA-MB-231 cells. We also confirmed that the increased expression of PD-L1 mediated by extracellular acidosis was decreased when the pH was raised to the normal range. Gene set enrichment analysis (GSEA) of public breast cancer patient databases showed that PD-L1 expression was also highly correlated with IL-6/JAK/STAT3 signaling. Surprisingly, the expression of both phospho-tyrosine STAT3 and PD-L1 was significantly increased under conditions of extracellular acidosis, and inhibition of STAT3 did not increase the expression of PD-L1 even under acidic conditions in MDA-MB-231 cells. Based on these results, we suggest that the expression of PD-L1 is increased by tumor acidosis via activation of STAT3 in MDA-MB-231 cells.

STAT3 activation in microglia exacerbates hippocampal neuronal apoptosis in diabetic brains.

Diabetes mellitus (DM) characterized by hyperglycemia leads to a variety of complications, including cognitive impairment or memory loss. The hippocampus is a key brain area for learning and memory and is one of the regions that is most sensitive to diabetes. However, the pathogenesis of diabetic neuronal lesion is not yet completely understood. We focused on the association of microglia activation and brain lesions in diabetes. In this study, we investigated whether and how signal transducer and activator of transcription 3 (STAT3) activation in microglia affects neuronal lesions in diabetic brains. Using a streptozotocin-induced type 1 DM model, we showed enhanced hippocampal neuronal apoptosis that was associated with increased STAT3 activation. We found that hyperglycemia increased the expression of inflammatory cytokines such as interferon-γ (IFN-γ) and interleukin-6, in the diabetic hippocampus. In particular, IFN-γ induced autocrine activation of microglia, and STAT3 activation is important for this process. We also demonstrated that STAT3 activation in microglia increased tumor necrosis factor-α (TNF-α) expression; subsequently, TNF-α increased neuronal apoptosis by increasing reactive oxygen species (ROS) levels in the neuronal cells. We also took advantage of mice lacking STAT3 in microglia and demonstrated that depletion of microglial STAT3 reduced neuronal apoptosis in the diabetic hippocampus. Taken together, these results suggest that STAT3 activation in microglia plays an important role in hyperglycemia-induced neuronal apoptosis in the diabetic hippocampus and provide a potential therapeutic benefit of STAT3 inhibition in microglia for preventing diabetic neuronal lesions.

Phylogenetic analysis of ABCG subfamily proteins in plants: functional clustering and coevolution with ABCGs of pathogens.

ABCG subfamily proteins are highly enriched in terrestrial plants. Many of these proteins secrete secondary metabolites that repel or inhibit pathogens. To establish why the ABCG subfamily proteins proliferated extensively during evolution, we constructed phylogenetic trees from a broad range of eukaryotic organisms. ABCG proteins were massively duplicated in land plants and in oomycetes, a group of agronomically important plant pathogens, which prompted us to hypothesize that plant and pathogen ABCGs coevolved. Supporting this hypothesis, full-size ABCGs in host plants (Arabidopsis thaliana and Glycine max) and their pathogens (Hyaloperonospora arabidopsidis and Phytophthora sojae, respectively) had similar divergence times and patterns. Furthermore, generalist pathogens with broad ranges of host plants have diversified more ABCGs than their specialist counterparts. The hypothesis was further tested using an example pair of ABCGs that first diverged during multiplication in a host plant and its pathogen: AtABCG31 of A. thaliana and HpaP802307 of H. arabidopsidis. AtABCG31 expression was activated following infection with H. arabidopsidis, and disrupting AtABCG31 led to increased susceptibility to H. arabidopsidis. Together, our results suggest that ABCG genes in plants and their oomycete pathogens coevolved in an arms race, to extrude secondary metabolites involved in the plant's defense response against pathogens.

Correction to: Potential causes and consequences of rapid mitochondrial genome evolution in thermoacidophilic Galdieria (Rhodophyta).

An amendment to this paper has been published and can be accessed via the original article.

Potential causes and consequences of rapid mitochondrial genome evolution in thermoacidophilic Galdieria (Rhodophyta).

BACKGROUND: The Cyanidiophyceae is an early-diverged red algal class that thrives in extreme conditions around acidic hot springs. Although this lineage has been highlighted as a model for understanding the biology of extremophilic eukaryotes, little is known about the molecular evolution of their mitochondrial genomes (mitogenomes). RESULTS: To fill this knowledge gap, we sequenced five mitogenomes from representative clades of Cyanidiophyceae and identified two major groups, here referred to as Galdieria-type (G-type) and Cyanidium-type (C-type). G-type mitogenomes exhibit the following three features: (i) reduction in genome size and gene inventory, (ii) evolution of unique protein properties including charge, hydropathy, stability, amino acid composition, and protein size, and (iii) distinctive GC-content and skewness of nucleotides. Based on GC-skew-associated characteristics, we postulate that unidirectional DNA replication may have resulted in the rapid evolution of G-type mitogenomes. CONCLUSIONS: The high divergence of G-type mitogenomes was likely driven by natural selection in the multiple extreme environments that Galdieria species inhabit combined with their highly flexible heterotrophic metabolism. We speculate that the interplay between mitogenome divergence and adaptation may help explain the dominance of Galdieria species in diverse extreme habitats.

Retinal pigment epithelium-derived transforming growth factor-ß2 inhibits the angiogenic response of endothelial cells by decreasing vascular endothelial growth factor receptor-2 expression.

Transforming growth factor-ß (TGF-ß) is a multifunctional cytokine that is known to modulate various aspects of endothelial cell (EC) biology. Retinal pigment epithelium (RPE) is important for regulating angiogenesis of choriocapillaris and one of the main cell sources of TGF-ß secretion, particularly TGF-ß2. However, it is largely unclear whether and how TGF-ß2 affects angiogenic responses of ECs. In the current study, we demonstrated that TGF-ß2 reduces vascular endothelial growth factor receptor-2 (VEGFR-2) expression in ECs and thereby inhibits vascular endothelial growth factor (VEGF) signaling and VEGF-induced angiogenic responses such as EC migration and tube formation. We also demonstrated that the reduction of VEGFR-2 expression by TGF-ß2 is due to the suppression of JNK signaling. In coculture of RPE cells and ECs, RPE cells decreased VEGFR-2 levels in ECs and EC migration. In addition, we showed that TGF-ß2 derived from RPE cells is involved in the reduction of VEGFR-2 expression and inhibition of EC migration. These results suggest that TGF-ß2 plays an important role in inhibiting the angiogenic responses of ECs during the interaction between RPE cells and ECs and that angiogenic responses of ECs may be amplified by a decrease in TGF-ß2 expression in RPE cells under pathologic conditions.

Interaction between microglia and retinal pigment epithelial cells determines the integrity of outer blood-retinal barrier in diabetic retinopathy.

Inner and outer blood-retinal barriers (BRBs), mainly composed of retinal endothelial cells and retinal pigment epithelial (RPE) cells, respectively, maintain the integrity of the retinal tissues. In this study, we aimed to investigate the mechanisms of the outer BRB disruption regarding the interaction between RPE and microglia. In mice with high-fat diet-induced obesity and streptozotocin-induced hyperglycemia, microglia accumulated on the RPE layer, as in those after intravitreal injection of interleukin (IL)-6, which is elevated in ocular fluids of patients with diabetic retinopathy. Although IL-6 did not directly affect the levels of zonula occludens (ZO)-1 and occludin in RPE cells, IL-6 increased VEGFA mRNA in RPE cells to recruit microglial cells. In microglial cells, IL-6 upregulated the mRNA levels of MCP1, MIP1A, and MIP1B, to amplify the recruitment of microglial cells. In this manner, IL-6 modulated RPE and microglial cells to attract microglial cells on RPE cells. Furthermore, IL-6-treated microglial cells produced and secreted tumor necrosis factor (TNF)-α, which activated NF-κB and decreased the levels of ZO-1 in RPE cells. As STAT3 inhibition reversed the effects of IL-6-treated microglial cells on the RPE monolayer in vitro, it reduced the recruitment of microglial cells and the production of TNF-α in RPE tissues in streptozotocin-treated mice. Taken together, IL-6-treated RPE and microglial cells amplified the recruitment of microglial cells and IL-6-treated microglial cells produced TNF-α to disrupt the outer BRB in diabetic retinopathy.

Angiopoietin 1 attenuates interleukin-6-induced endothelial cell permeability through SHP-1.

The regulation of endothelial cell (EC) permeability is critical for the physiological homeostasis of blood vessels and tissues. The elevation of pro-inflammatory cytokines is highly associated with lesions, such as the increased vascular permeability of diabetic retinas. We have previously reported that interleukin-6 (IL-6) increases EC permeability through the downregulation of tight junction protein expression. Angiopoietin 1 (Ang1) has an anti-permeability function, but the effect of Ang1 on vascular permeability induced by inflammatory cytokines is unclear. In the present study, we investigated the effect of Ang1 on IL-6-induced EC permeability and its underlying molecular mechanisms. We demonstrated that Ang1 inhibited the IL-6-induced increase in EC permeability by inhibiting the reductions in the levels of tight junction protein ZO-1 and occludin, which was related to the decrease in vascular endothelial growth factor (VEGF) secretion through the inhibition of STAT3 activation by Ang1. Mechanistically, Ang1 induced the dissociation of the tyrosine phosphatase SHP-1 from the Tie2 receptor and increased the binding of SHP-1 to JAK1, JAK2, and STAT3, which are IL-6 downstream signaling proteins. We conclude that SHP-1 plays an important role in the Ang1-induced inhibition of JAK/STAT3 signaling. These results provide evidence for a potential beneficial role of Ang1 in suppressing the vascular permeability induced by the pro-inflammatory cytokine IL-6 in diabetic retinopathy.

ß-Adrenergic receptor agonists attenuate pericyte loss in diabetic retinas through Akt activation.

Pericytes (PCs) are crucial in maintaining the quiescence of endothelial cells (ECs) and the integrity of EC tight junctions. Especially in diabetic retinopathy (DR), PC loss is one of the early pathologic changes in capillaries of diabetic retinas. Thus, preventing PC loss is beneficial for attenuating vision impairment in patients with DR. Although many studies have revealed the mechanism of PC loss in retinas, little is known about the mechanisms that increase PC survival. We focused on the effect of ß-adrenergic receptor agonists (ß-agonists) on PC loss in diabetic retinas. In this study, ß-agonists increased the cell viability of PCs by increasing PC survival and proliferation. Mechanistically, ß-agonist-induced protein kinase B activation in PCs reduced PC apoptosis in response to various stimuli. ß2-agonists more potently increased PC survival than ß1-agonists. ß2-Agonist reduced vascular leakage and PC loss in retinas of mice with streptozotocin-induced diabetes. In cocultures of PCs and ECs, ß2-agonists restored the altered permeability and ZO-1 expression in ECs induced by PC loss. We concluded that ß-agonists, especially ß2-agonists, increase PC survival, thereby preventing diabetes-induced PC loss in retinas. These results provide a potential therapeutic benefit of ß-agonists for preventing PC loss in DR.-Yun, J.-H., Jeong, H.-S., Kim, K.-J., Han, M. H., Lee, E. H., Lee, K., Cho, C.-H. ß-Adrenergic receptor agonists attenuate pericyte loss in diabetic retinas through Akt activation.

Propranolol increases vascular permeability through pericyte apoptosis and exacerbates oxygen-induced retinopathy.

Retinopathy of prematurity (ROP) is an eye disease that causes blindness due to delayed vascular growth, retinal ischemia, and resulting abnormal angiogenesis. Nonselective ß-antagonist propranolol is in clinical trials for the treatment of ROP due to its effect of reducing VEGF expression and inhibiting retinal angiogenesis in oxygen-induced ROP models (OIR), but the mechanism by which propranolol acts on ROP vessels is still unclear. In the present study, we have focused on the effect of propranolol on pericyte survival and vascular permeability. We demonstrated that propranolol increases pericyte apoptosis more sensitively than endothelial cells (ECs), thereby weakening EC tight junctions to increase endothelial permeability in co-cultures of pericytes and ECs. Mechanistically, pericyte apoptosis by propranolol was due to the inhibition of Akt signaling pathway. We also demonstrated that propranolol increases pericyte loss and vascular permeability of retinal vessels in a mouse model of OIR. These results suggest that propranolol may be negative for blood vessels in retinas of OIR, and that the efficacy of propranolol for the treatment of ROP needs to be more thoroughly verified.

Endothelial STAT3 Activation Increases Vascular Leakage Through Downregulating Tight Junction Proteins: Implications for Diabetic Retinopathy.

Vascular inflammation is characteristic feature of diabetic retinopathy. In diabetic retina, a variety of the pro-inflammatory cytokines are elevated and involved in endothelial dysfunction. STAT3 transcription factor has been implicated in mediating cytokine signaling during vascular inflammation. However, whether and how STAT3 is involved in the direct regulation of the endothelial permeability is currently undefined. Our studies revealed that IL-6-induced STAT3 activation increases retinal endothelial permeability and vascular leakage in retinas of mice through the reduced expression of the tight junction proteins ZO-1 and occludin. In a co-culture model with microglia and endothelial cells under a high glucose condition, the microglia-derived IL-6 induced STAT3 activation in the retinal endothelial cells, leading to increasing endothelial permeability. In addition, IL-6-induced STAT3 activation was independent of ROS generation in the retinal endothelial cells. Moreover, we demonstrated that STAT3 activation downregulates the ZO-1 and occludin levels and increases the endothelial permeability through the induction of VEGF production in retinal endothelial cells. These results suggest the potential importance of IL-6/STAT3 signaling in regulating endothelial permeability and provide a therapeutic target to prevent the pathology of diabetic retinopathy. J. Cell. Physiol. 232: 1123-1134, 2017. © 2016 Wiley Periodicals, Inc.

Parallel evolution of highly conserved plastid genome architecture in red seaweeds and seed plants.

BACKGROUND: The red algae (Rhodophyta) diverged from the green algae and plants (Viridiplantae) over one billion years ago within the kingdom Archaeplastida. These photosynthetic lineages provide an ideal model to study plastid genome reduction in deep time. To this end, we assembled a large dataset of the plastid genomes that were available, including 48 from the red algae (17 complete and three partial genomes produced for this analysis) to elucidate the evolutionary history of these organelles. RESULTS: We found extreme conservation of plastid genome architecture in the major lineages of the multicellular Florideophyceae red algae. Only three minor structural types were detected in this group, which are explained by recombination events of the duplicated rDNA operons. A similar high level of structural conservation (although with different gene content) was found in seed plants. Three major plastid genome architectures were identified in representatives of 46 orders of angiosperms and three orders of gymnosperms. CONCLUSIONS: Our results provide a comprehensive account of plastid gene loss and rearrangement events involving genome architecture within Archaeplastida and lead to one over-arching conclusion: from an ancestral pool of highly rearranged plastid genomes in red and green algae, the aquatic (Florideophyceae) and terrestrial (seed plants) multicellular lineages display high conservation in plastid genome architecture. This phenomenon correlates with, and could be explained by, the independent and widely divergent (separated by >400 million years) origins of complex sexual cycles and reproductive structures that led to the rapid diversification of these lineages.

ITF2 prevents activation of the ß-catenin-TCF4 complex in colon cancer cells and levels decrease with tumor progression.

BACKGROUND & AIMS: Immunoglobulin transcription factor 2 (ITF2) was believed to promote neoplastic transformation via activation of ß-catenin. However, ITF2 recently was reported to suppress colon carcinogenesis. We investigated the roles of ITF2 in colorectal cancer cell lines and tumor formation and growth in mice. METHODS: Levels of ITF2, ß-catenin, and c-Myc were measured in 12 human colorectal tumor samples and by immunohistochemistry. ITF2 regulation of ß-catenin and T-cell factor (TCF) were analyzed using luciferase reporter, reverse-transcription quantitative polymerase chain reaction, flow cytometry, and immunoblot analyses. Mice were given subcutaneous injections of human colorectal cancer cell lines that stably express ITF2, small hairpin RNAs to reduce levels of ITF2, or control plasmids; xenograft tumor growth was assessed. Human colorectal carcinoma tissue arrays were used to associate levels of ITF2 expression and clinical outcomes. RESULTS: Levels of ß-catenin, cMyc, and ITF2 were increased in areas of human colon adenomas and carcinomas, compared with nontumor areas of the same tissues. ITF2 levels were reduced and cMyc levels were increased in areas of carcinoma, compared with adenoma. In human colorectal cancer cell lines, activation of the ß-catenin-TCF4 complex and expression of its target genes were regulated negatively by ITF2. ITF2 inhibited formation of the ß-catenin-TCF4 complex by competing with TCF4 for ß-catenin binding. Stable transgenic expression of ITF2 in human colorectal cancer cell lines reduced their proliferation and tumorigenic potential in mice, whereas small hairpin RNA knockdown of ITF2 promoted growth of xenograft tumors in mice. In an analysis of colorectal tumor tissue arrays, loss of ITF2 from colorectal tumor tissues was associated with poor outcomes of patients. A gene set enrichment analysis supported the negative correlation between the level of ITF2 and activity of the ß-catenin-TCF4 complex. CONCLUSIONS: In human colorectal cancer cell lines and tissue samples, ITF2 appears to prevent activation of the ß-catenin-TCF4 complex and transcription of its gene targets. Loss of ITF2 promotes the ability of colorectal cancer cells to form xenograft tumors, and is associated with tumor progression and shorter survival times of patients.

Interaction between mitsugumin 29 and TRPC3 participates in regulating Ca(2+) transients in skeletal muscle.

Mitsugumin 29 (MG29) is related to the fatigue and aging processes of skeletal muscle. To examine the roles of MG29 in conjunction with its binding protein, the canonical-type transient receptor potential cation channel 3 (TRPC3), in skeletal muscle, the binding region of MG29 to TRPC3 was studied along with the functional relevance of the binding in mouse primary skeletal myotubes using co-immunoprecipitation assays and Ca(2+) imaging experiments. The N-terminus and the I-II loop of MG29 constitute the binding region for TRPC3. The myotubes that expressed the MG29 mutant missing the entire TRPC3-binding region showed a disrupted binding between endogenous MG29 and TRPC3 and a reduction in Ca(2+) transients in response to membrane depolarization without affecting ryanodine receptor 1 activity, the resting cytosolic Ca(2+) level, and the amount of releasable Ca(2+) from the sarcoplasmic reticulum. Among the proteins mediating Ca(2+) movements in skeletal muscle, TRPC4 expression was significantly decreased by the MG29 mutant. Therefore, MG29 could be a new factor for regulating Ca(2+) transients during skeletal muscle contraction possibly via a correlation with TRPC3 and TRPC4.