FOG2 Protein Down-regulation by Transforming Growth Factor-β1-induced MicroRNA-200b/c Leads to Akt Kinase Activation and Glomerular Mesangial Hypertrophy Related to Diabetic Nephropathy

Glomerular hypertrophy is a hallmark of diabetic nephropathy. Akt kinase activated by transforming growth factor-β1 (TGF-β) plays an important role in glomerular mesangial hypertrophy. However, the mechanisms of Akt activation by TGF-β are not fully understood. Recently, miR-200 and its target FOG2 were reported to regulate the activity of phosphatidylinositol 3-kinase (the upstream activator of Akt) in insulin signaling. Here, we show that TGF-β activates Akt in glomerular mesangial cells by inducing miR-200b and miR-200c, both of which target FOG2, an inhibitor of phosphatidylinositol 3-kinase activation. FOG2 expression was reduced in the glomeruli of diabetic mice as well as TGF-β-treated mouse mesangial cells (MMC). FOG2 knockdown by siRNAs in MMC activated Akt and increased the protein content/cell ratio suggesting hypertrophy. A significant increase of miR-200b/c levels was detected in diabetic mouse glomeruli and TGF-β-treated MMC. Transfection of MMC with miR-200b/c mimics significantly decreased the expression of FOG2. Conversely, miR-200b/c inhibitors attenuated TGF-β-induced decrease in FOG2 expression. Furthermore, miR-200b/c mimics increased the protein content/cell ratio, whereas miR-200b/c inhibitors abrogated the TGF-β-induced increase in protein content/cell. In addition, down-regulation of FOG2 by miR-200b/c could activate not only Akt but also ERK, which was also through PI3K activation. These data suggest a new mechanism for TGF-β-induced Akt activation through FOG2 down-regulation by miR-200b/c, which can lead to glomerular mesangial hypertrophy in the progression of diabetic nephropathy.     

SCFFbxw15 Mediates Histone Acetyltransferase Binding to Origin Recognition Complex (HBO1) Ubiquitin-Proteasomal Degradation to Regulate Cell Proliferation

Histone acetyltransferase binding to origin recognition complex (HBO1) plays a crucial role in DNA replication licensing and cell proliferation, yet its molecular regulation in cells is relatively unknown. Here an uncharacterized protein, Fbxw15, directly interacts with HBO1, a labile protein (t_½ = ∼3 h), to mediate its ubiquitination (Lys³³⁸) and degradation in the cytoplasm. Fbxw15-mediated HBO1 depletion required mitogen-activated protein kinase 1 (Mek1), which was sufficient to trigger HBO1 phosphorylation and degradation in cells. Mek1 ability to produce HBO1 degradation was blocked by Fbxw15 silencing. Lipopolysaccharide induced HBO1 degradation, an effect abrogated by Fbxw15 or Mek1 cellular depletion. Modulation of Fbxw15 levels was able to differentially regulate histone H3K14 acetylation and cellular proliferation by altering HBO1 levels. These studies authenticate Fbxw15 as a ubiquitin E3 ligase subunit that mediates endotoxin-induced HBO1 depletion in cells, thereby controlling cell replicative capacity.     

Effect of Feeding Millet (Sorghum vulgarie) Protein at Various Protein Levels on Adrenal Lipids of Rats

A significant increase in adrenal weight, total lipids, cholesterol phospholipids and glycerides (mono-and triglycerides) was observed in rats fed millet at 5, 10 and 15 % protein levels respectively for a period of six weeks as compared to rats fed casein at 10 per cent level. Increases in cholesterol were in both its free and esterified fraction. Adrenal phosphatidyl etha-nolamine was increased in all millet fed rats whereas phosphatidyl choline increased in M–15 % and decreased in M–5 % groups. Other phospholipid fractions viz. monophosphatidyl inositol, lysophosphatidyl ethanolamine, sphingomyeline, phosphatidic acid and polyglycerophosphatide also showed significant alterations in rats fed millet protein as compared to control. Incorporation of acetate–l–¹⁴C into adrenal lipids was lower and that of glucose–U–¹⁴C, palmitate–l–¹⁴C and NaH₂³²PO₄ was higher than the control.     

Induction of IL-10 and TGFβ from CD4+CD25+FoxP3+ T Cells Correlates with Parasite Load in Indian Kala-azar Patients Infected with Leishmania donovani

Background

Visceral leishmaniasis (VL) is distinguished by a complex interplay of immune response and parasite multiplication inside host cells. However, the direct association between different immunological correlates and parasite numbers remains largely unknown.

Methodology/Principal Findings

We examined the plasma levels of different disease promoting/protective as well as Th17 cytokines and found IL-10, TGFβ and IL-17 to be significantly correlated with parasite load in VL patients (r = 0.52, 0.53 and 0.51 for IL-10, TGFβ and IL-17, respectively). We then extended our investigation to a more antigen-specific response and found leishmanial antigen stimulated levels of both IL-10 and TGFβ to be significantly associated with parasite load (r = 0.71 and 0.72 for IL-10 and TGFβ respectively). In addition to cytokines we also looked for different cellular subtypes that could contribute to cytokine secretion and parasite persistence. Our observations manifested an association between different Treg cell markers and disease progression as absolute numbers of CD4⁺CD25⁺ (r = 0.55), CD4⁺CD25^hi (r = 0.61) as well as percentages of CD4⁺CD25⁺FoxP3⁺ T cells (r = 0.68) all correlated with parasite load. Encouraged by these results, we investigated a link between these immunological components and interestingly found both CD4⁺CD25⁺ and CD4⁺CD25⁺FoxP3⁺ Treg cells to secrete significantly (p<0.05) higher amounts of not only IL-10 but also TGFβ in comparison to corresponding CD25^- T cells.

Conclusions/Significance

Our findings shed some light on source(s) of TGFβ and suggest an association between these disease promoting cytokines and Treg cells with parasite load during active disease. Moreover, the direct evidence of CD4⁺CD25⁺FoxP3⁺ Treg cells as a source of IL-10 and TGFβ during active VL could open new avenues for immunotherapy towards cure of this potentially fatal disease.     

DNAH6 and Its Interactions with PCD Genes in Heterotaxy and Primary Ciliary Dyskinesia

Heterotaxy, a birth defect involving left-right patterning defects, and primary ciliary dyskinesia (PCD), a sinopulmonary disease with dyskinetic/immotile cilia in the airway are seemingly disparate diseases. However, they have an overlapping genetic etiology involving mutations in cilia genes, a reflection of the common requirement for motile cilia in left-right patterning and airway clearance. While PCD is a monogenic recessive disorder, heterotaxy has a more complex, largely non-monogenic etiology. In this study, we show mutations in the novel dynein gene DNAH6 can cause heterotaxy and ciliary dysfunction similar to PCD. We provide the first evidence that trans-heterozygous interactions between DNAH6 and other PCD genes potentially can cause heterotaxy. DNAH6 was initially identified as a candidate heterotaxy/PCD gene by filtering exome-sequencing data from 25 heterotaxy patients stratified by whether they have airway motile cilia defects. dnah6 morpholino knockdown in zebrafish disrupted motile cilia in Kupffer’s vesicle required for left-right patterning and caused heterotaxy with abnormal cardiac/gut looping. Similarly DNAH6 shRNA knockdown disrupted motile cilia in human and mouse respiratory epithelia. Notably a heterotaxy patient harboring heterozygous DNAH6 mutation was identified to also carry a rare heterozygous PCD-causing DNAI1 mutation, suggesting a DNAH6/DNAI1 trans-heterozygous interaction. Furthermore, sequencing of 149 additional heterotaxy patients showed 5 of 6 patients with heterozygous DNAH6 mutations also had heterozygous mutations in DNAH5 or other PCD genes. We functionally assayed for DNAH6/DNAH5 and DNAH6/DNAI1 trans-heterozygous interactions using subthreshold double-morpholino knockdown in zebrafish and showed this caused heterotaxy. Similarly, subthreshold siRNA knockdown of Dnah6 in heterozygous Dnah5 or Dnai1 mutant mouse respiratory epithelia disrupted motile cilia function. Together, these findings support an oligogenic disease model with broad relevance for further interrogating the genetic etiology of human ciliopathies.     

Formin-mediated actin polymerization at cell–cell junctions stabilizes E-cadherin and maintains monolayer integrity during wound repair

Cadherin-mediated cell–cell adhesion is required for epithelial tissue integrity in homeostasis, during development, and in tissue repair. E-cadherin stability depends on F-actin, but the mechanisms regulating actin polymerization at cell–cell junctions remain poorly understood. Here we investigated a role for formin-mediated actin polymerization at cell–cell junctions. We identify mDia1 and Fmnl3 as major factors enhancing actin polymerization and stabilizing E-cadherin at epithelial junctions. Fmnl3 localizes to adherens junctions downstream of Src and Cdc42 and its depletion leads to a reduction in F-actin and E-cadherin at junctions and a weakening of cell–cell adhesion. Of importance, Fmnl3 expression is up-regulated and junctional localization increases during collective cell migration. Depletion of Fmnl3 or mDia1 in migrating monolayers results in dissociation of leader cells and impaired wound repair. In summary, our results show that formin activity at epithelial cell–cell junctions is important for adhesion and the maintenance of epithelial cohesion during dynamic processes, such as wound repair.     

Discovery of covalent enzyme inhibitors using virtual docking of covalent fragments

Here we present a virtual docking screen of 1648 commercially available covalent fragments, and identified covalent inhibitors of cysteine protease cathepsin L. These inhibitors did not inhibit closely related protease cathepsin B. Thus, we have established virtual docking of covalent fragments as an approach to discover covalent enzyme inhibitors.