An inactivating human TRPC6 channel mutation without focal segmental glomerulosclerosis.

Transient receptor potential cation channel-6 (TRPC6) gene mutations cause familial focal segmental glomerulosclerosis (FSGS), which is inherited as an autosomal dominant disease. In patients with TRPC6-related FSGS, all mutations map to the N- or C-terminal TRPC6 protein domains. Thus far, the majority of TRPC6 mutations are missense resulting in increased or decreased calcium influx; however, the fundamental molecular mechanisms causing cell injury and kidney pathology are unclear. We report a novel heterozygous TRPC6 mutation (V691Kfs*) in a large kindred with no signs of FSGS despite a largely truncated TRPC6 protein. We studied the molecular effects of V691Kfs* TRPC6 mutant using the tridimensional cryo-EM structure of the tetrameric TRPC6 protein. The results indicated that V691 is localized at the pore-forming transmembrane region affecting the ion conduction pathway, and predicted that V691Kfs* causes closure of the ion-conducting pathway leading to channel inactivation. We assessed the impact of V691Kfs* and two previously reported TRPC6 disease mutants (P112Q and G757D) on calcium influx in cells. Our data show that the V691Kfs* fully inactivated the TRCP6 channel-specific calcium influx consistent with a complete loss-of-function phenotype. Furthermore, the V691Kfs* truncation exerted a dominant negative effect on the full-length TRPC6 proteins. In conclusion, the V691Kfs* non-functional truncated TRPC6 is not sufficient to cause FSGS. Our data corroborate recently characterized TRPC6 loss-of-function and gain-of-function mutants suggesting that one defective TRPC6 gene copy is not sufficient to cause FSGS. We underscore the importance of increased rather than reduced calcium influx through TRPC6 for podocyte cell death.

Structural expression of bovine milk and meat factors in tissues of colorectal, lung and pancreatic cancer patients.

Chronic inflammation, linked to the presence of bovine milk and meat factors (BMMFs) and specific subsets of macrophages, results in oxygen radical synthesis and induction of mutations in DNA of actively replicating cells and replicating single stranded DNA. Cancers arising from this process have been characterized as indirect carcinogenesis by infectious agents (without persistence of genes of the agent in premalignant or cancers cells). Here, we investigate structural properties of pleomorphic vesicles, regularly identified by staining peritumor tissues of colorectal, lung and pancreatic cancer for expression of BMMF Rep. The latter represents a subgroup of BMMF1 proteins involved in replication of small single-stranded circular plasmids of BMMF, but most likely also contributing to pleomorphic vesicular structures found in the periphery of colorectal, lung and pancreatic cancers. Structurally dense regions are demonstrated in preselected areas of colorectal cancer, after staining with monoclonal antibodies against BMMF1 Rep. Similar structures were observed in human embryonic cells (HEK293TT) overexpressing Rep. These data suggest that Rep or Rep isoforms contribute to the structural formation of vesicles.

Blockade of Glycosphingolipid Synthesis Inhibits Cell Cycle and Spheroid Growth of Colon Cancer Cells In Vitro and Experimental Colon Cancer Incidence In Vivo.

Colorectal cancer (CRC) is one of the most frequently diagnosed cancers in humans. At early stages CRC is treated by surgery and at advanced stages combined with chemotherapy. We examined here the potential effect of glucosylceramide synthase (GCS)-inhibition on CRC biology. GCS is the rate-limiting enzyme in the glycosphingolipid (GSL)-biosynthesis pathway and overexpressed in many human tumors. We suppressed GSL-biosynthesis using the GCS inhibitor Genz-123346 (Genz), NB-DNJ (Miglustat) or by genetic targeting of the GCS-encoding gene UDP-glucose-ceramide-glucosyltransferase- (UGCG). GCS-inhibition or GSL-depletion led to a marked arrest of the cell cycle in Lovo cells. UGCG silencing strongly also inhibited tumor spheroid growth in Lovo cells and moderately in HCT116 cells. MS/MS analysis demonstrated markedly elevated levels of sphingomyelin (SM) and phosphatidylcholine (PC) that occurred in a Genz-concentration dependent manner. Ultrastructural analysis of Genz-treated cells indicated multi-lamellar lipid storage in vesicular compartments. In mice, Genz lowered the incidence of experimentally induced colorectal tumors and in particular the growth of colorectal adenomas. These results highlight the potential for GCS-based inhibition in the treatment of CRC.

Gangliosides modulate insulin secretion by pancreatic beta cells under glucose stress.

In pancreatic beta cells, the entry of glucose and downstream signaling for insulin release is regulated by the glucose transporter 2 (Glut2) in rodents. Dysfunction of the insulin-signaling cascade may lead to diabetes mellitus. Gangliosides, sialic acid-containing glycosphingolipids (GSLs), have been reported to modulate the function of several membrane proteins.Murine islets express predominantly sialylated GSLs, particularly the simple gangliosides GM3 and GD3 having a potential modulatory role in Glut2 activity. Conditional, tamoxifen-inducible gene targeting in pancreatic islets has now shown that mice lacking the glucosylceramide synthase (Ugcg), which represents the rate-limiting enzyme in GSL biosynthesis, displayed impaired glucose uptake and showed reduced insulin secretion. Consequently, mice with pancreatic GSL deficiency had higher blood glucose levels than respective controls after intraperitoneal glucose application. High-fat diet feeding enhanced this effect. GSL-deficient islets did not show apoptosis or ER stress and displayed a normal ultrastructure. Their insulin content, size and number were similar as in control islets. Isolated beta cells from GM3 synthase null mice unable to synthesize GM3 and GD3 also showed lower glucose uptake than respective control cells, corroborating the results obtained from the cell-specific model. We conclude that in particular the negatively charged gangliosides GM3 and GD3 of beta cells positively influence Glut2 function to adequately respond to high glucose loads.

Immunosuppression and Aberrant T Cell Development in the Absence of N-Myristoylation.

N-myristoylation refers to the attachment of myristic acid to the N-terminal glycine of proteins and substantially affects their intracellular targeting and functions. The thymus represents an organ with a prominent N-myristoylation activity. To elucidate the role of protein N-myristoylation for thymocyte development, we generated mice with a T cell lineage-specific deficiency in N-myristoyl transferase (Nmt)1 and 2. Depletion of Nmt activity in T cells led to a defective transmission of TCR signals, a developmental blockage of thymocytes at the transition from double-negative 3 to 4 stages, and a reduction of all the following stages. We could demonstrate that Lck and myristoylated alanine-rich C kinase substrate, two main myristoylated kinases in T cells, were mislocalized in the absence of Nmt activity. N-myristoylation was also indispensable for early and distal TCR signaling events such as CD3ζ, Zap70, and Erk activation and for release of cytokines such as IFN-γ and IL-2. As a consequence, the initiation and propagation of the TCR signaling cascade was severely impaired. Furthermore, we showed that the absence of myristoylation had an immunosuppressive effect on T cells in vivo after treatment with CpG and stimulation of the TCR with the staphylococcal enterotoxin B superantigen. Therefore, protein myristoylation is indispensable in T cell development and activation and its inhibition might offer a novel strategy to achieve immunosuppression.

Zeb1 affects epithelial cell adhesion by diverting glycosphingolipid metabolism.

This study proposes that the transcription factor Zeb1 modulates epithelial cell adhesion by diverting glycosphingolipid metabolism. Zeb1 promotes expression of a-series glycosphingolipids via regulating expression of GM3 synthase (St3gal5), which mechanistically involves Zeb1 binding to the St3gal5 promoter as well as suppressing microRNA-mediated repression of St3gal5. Functionally, the repression of St3gal5 suffices to elevate intercellular adhesion and expression of distinct junction-associated proteins, reminiscent of knockdown of Zeb1. Conversely, overexpressing St3gal5 sensitizes cells towards TGF-ß1-induced disruption of cell-cell interaction and partially antagonizes elevation of intercellular adhesion imposed by Zeb1 knockdown. These results highlight a direct connection of glycosphingolipid metabolism and epithelial cell adhesion via Zeb1.

Histone deacetylase 10 promotes autophagy-mediated cell survival.

Tumor cells activate autophagy in response to chemotherapy-induced DNA damage as a survival program to cope with metabolic stress. Here, we provide in vitro and in vivo evidence that histone deacetylase (HDAC)10 promotes autophagy-mediated survival in neuroblastoma cells. We show that both knockdown and inhibition of HDAC10 effectively disrupted autophagy associated with sensitization to cytotoxic drug treatment in a panel of highly malignant V-MYC myelocytomatosis viral-related oncogene, neuroblastoma derived-amplified neuroblastoma cell lines, in contrast to nontransformed cells. HDAC10 depletion in neuroblastoma cells interrupted autophagic flux and induced accumulation of autophagosomes, lysosomes, and a prominent substrate of the autophagic degradation pathway, p62/sequestosome 1. Enforced HDAC10 expression protected neuroblastoma cells against doxorubicin treatment through interaction with heat shock protein 70 family proteins, causing their deacetylation. Conversely, heat shock protein 70/heat shock cognate 70 was acetylated in HDAC10-depleted cells. HDAC10 expression levels in high-risk neuroblastomas correlated with autophagy in gene-set analysis and predicted treatment success in patients with advanced stage 4 neuroblastomas. Our results demonstrate that HDAC10 protects cancer cells from cytotoxic agents by mediating autophagy and identify this HDAC isozyme as a druggable regulator of advanced-stage tumor cell survival. Moreover, these results propose a promising way to considerably improve treatment response in the neuroblastoma patient subgroup with the poorest outcome.

Neuronal expression of glucosylceramide synthase in central nervous system regulates body weight and energy homeostasis.

Hypothalamic neurons are main regulators of energy homeostasis. Neuronal function essentially depends on plasma membrane-located gangliosides. The present work demonstrates that hypothalamic integration of metabolic signals requires neuronal expression of glucosylceramide synthase (GCS; UDP-glucose:ceramide glucosyltransferase). As a major mechanism of central nervous system (CNS) metabolic control, we demonstrate that GCS-derived gangliosides interacting with leptin receptors (ObR) in the neuronal membrane modulate leptin-stimulated formation of signaling metabolites in hypothalamic neurons. Furthermore, ganglioside-depleted hypothalamic neurons fail to adapt their activity (c-Fos) in response to alterations in peripheral energy signals. Consequently, mice with inducible forebrain neuron-specific deletion of the UDP-glucose:ceramide glucosyltransferase gene (Ugcg) display obesity, hypothermia, and lower sympathetic activity. Recombinant adeno-associated virus (rAAV)-mediated Ugcg delivery to the arcuate nucleus (Arc) significantly ameliorated obesity, specifying gangliosides as seminal components for hypothalamic regulation of body energy homeostasis.

Glycosphingolipids are essential for intestinal endocytic function.

Glycosphingolipids (GSLs) constitute major components of enterocytes and were hypothesized to be potentially important for intestinal epithelial polarization. The enzyme UDP-glucose ceramide glucosyltransferase (Ugcg) catalyzes the initial step of GSL biosynthesis. Newborn and adult mice with enterocyte-specific genetic deletion of the gene Ugcg were generated. In newborn mutants lacking GSLs at day P0, intestinal epithelia were indistinguishable from those in control littermates displaying an intact polarization with regular brush border. However, those mice were not consistently able to absorb nutritional lipids from milk. Between postnatal days 5 and 7, severe defects in intestinal epithelial differentiation occurred accompanied by impaired intestinal uptake of nutrients. Villi of mutant mice became stunted, and enterocytes lacked brush border. The defects observed in mutant mice caused diarrhea, malabsorption, and early death. In this study, we show that GSLs are essential for enterocyte resorptive function but are primarily not for polarization; GSLs are required for intracellular vesicular transport in resorption-active intestine.

Glucocorticoid activity during lung maturation is essential in mesenchymal and less in alveolar epithelial cells.

Corticosteroid treatment is an established therapy for preterm infants, and germline inactivation of the glucocorticoid receptor (GR) gene in the mouse leads to respiratory failure and postnatal lethality. Although glucocorticoids have been thought to critically act in epithelial cells inducing the functional maturation of the lung, inactivation of the GR gene exclusively in the epithelium of the developing murine lung did not impair survival. In contrast, mice lacking GR specifically in mesenchyme-derived cells displayed a phenotype strongly reminiscent of GR knockout animals and died immediately after birth. Detailed analysis of gene expression allows the conclusion that GR acts in cells of the fibroblast lineage controlling their proliferation rate and the composition of the extracellular matrix.

Iron regulatory proteins secure mitochondrial iron sufficiency and function.

Mitochondria supply cells with ATP, heme, and iron sulfur clusters (ISC), and mitochondrial energy metabolism involves both heme- and ISC-dependent enzymes. Here, we show that mitochondrial iron supply and function require iron regulatory proteins (IRP), cytosolic RNA-binding proteins that control mRNA translation and stability. Mice lacking both IRP1 and IRP2 in their hepatocytes suffer from mitochondrial iron deficiency and dysfunction associated with alterations of the ISC and heme biosynthetic pathways, leading to liver failure and death. These results uncover a major role of the IRPs in cell biology: to ensure adequate iron supply to the mitochondrion for proper function of this critical organelle.

Hepatic glycosphingolipid deficiency and liver function in mice.

UNLABELLED: Recent studies have reported that glycosphingolipids (GSLs) might be involved in obesity-induced insulin resistance. Those reports suggested that inhibition of GSL biosynthesis in animals ameliorated insulin resistance accompanied by improved glycemic control and decreased liver steatosis in obese mice. In addition, pharmacologic GSL depletion altered hepatic secretory function. In those studies, ubiquitously acting inhibitors for GSL biosynthesis have been used to inhibit the enzyme Ugcg (UDP-glucose:ceramide glucosyltransferase), catalyzing the first step of the glucosylceramide-based GSL-synthesis pathway. In the present study a genetic approach for selective GSL deletion in hepatocytes was chosen to achieve complete inhibition of GSL synthesis and to avoid possible adverse effects caused by Ugcg inhibitors. Using the Cre/loxP system under control of the albumin promoter, GSL biosynthesis in hepatocytes and their release into the plasma could be effectively blocked. Deletion of GSL in hepatocytes did not change the quantity of bile excretion through the biliary duct. Total bile salt content in bile, feces, and plasma from mutant mice showed no difference as compared to control animals. Cholesterol concentration in liver, bile, feces, and plasma samples remained unaffected. Lipoprotein concentrations in plasma samples in mutant animals reached similar levels as in their control littermates. No alteration in glucose tolerance after intraperitoneal application of glucose and insulin appeared in mutant animals. A preventive effect of GSL deficiency on development of liver steatosis after a high-fat diet was not observed. CONCLUSION: The data suggest that GSL in hepatocytes are not essential for sterol, glucose, or lipoprotein metabolism and do not prevent high-fat diet-induced liver steatosis, indicating that Ugcg inhibitors exert their effect on hepatocytes either independently of GSL or mediated by other (liver) cell types.

Kidney dendritic cell activation is required for progression of renal disease in a mouse model of glomerular injury.

The progression of kidney disease to renal failure correlates with infiltration of mononuclear immune cells into the tubulointerstitium. These infiltrates contain macrophages, DCs, and T cells, but the role of each cell type in disease progression is unclear. To investigate the underlying immune mechanisms, we generated transgenic mice that selectively expressed the model antigens ovalbumin and hen egg lysozyme in glomerular podocytes (NOH mice). Coinjection of ovalbumin-specific transgenic CD8+ CTLs and CD4+ Th cells into NOH mice resulted in periglomerular mononuclear infiltrates and inflammation of parietal epithelial cells, similar to lesions frequently observed in human chronic glomerulonephritis. Repetitive T cell injections aggravated infiltration and caused progression to structural and functional kidney damage after 4 weeks. Mechanistic analysis revealed that DCs in renal lymph nodes constitutively cross-presented ovalbumin and activated CTLs. These CTLs released further ovalbumin for CTL activation in the lymph nodes and for simultaneous presentation to Th cells by distinct DC subsets residing in the kidney tubulointerstitium. Crosstalk between tubulointerstitial DCs and Th cells resulted in intrarenal cytokine and chemokine production and in recruitment of more CTLs, monocyte-derived DCs, and macrophages. The importance of DCs was established by the fact that DC depletion rapidly resolved established kidney immunopathology. These findings demonstrate that glomerular antigen-specific CTLs and Th cells can jointly induce renal immunopathology and identify kidney DCs as a mechanistic link between glomerular injury and the progression of kidney disease.

E7 oncoprotein of novel human papillomavirus type 108 lacking the E6 gene induces dysplasia in organotypic keratinocyte cultures.

The genome organization of the novel human papillomavirus type 108 (HPV108), isolated from a low-grade cervical lesion, deviates from those of other HPVs in lacking an E6 gene. The three related HPV types HPV103, HPV108, and HPV101 were isolated from cervicovaginal cells taken from normal genital mucosa (HPV103) and low-grade (HPV108) and high-grade cervical (HPV101) intraepithelial neoplasia (Z. Chen, M. Schiffman, R. Herrero, R. DeSalle, and R. D. Burk, Virology 360:447-453, 2007, and this report). Their unusual genome organization, against the background of considerable phylogenetic distance from the other HPV types usually associated with lesions of the genital tract, prompted us to investigate whether HPV108 E7 per se is sufficient to induce the above-mentioned clinical lesions. Expression of HPV108 E7 in organotypic keratinocyte cultures increases proliferation and apoptosis, focal nuclear polymorphism, and polychromasia. This is associated with irregular intra- and extracellular lipid accumulation and loss of the epithelial barrier. These alterations are linked to HPV108 E7 binding to pRb and inducing its decrease, an increase in PCNA expression, and BrdU incorporation, as well as increased p53 and p21(CIP1) protein levels. A delay in keratin K10 expression, increased expression of keratins K14 and K16, and loss of the corneal proteins involucrin and loricrin have also been noted. These modifications are suggestive of infection by a high-risk papillomavirus.

Conditional inactivation of glucocorticoid receptor gene in dopamine-beta-hydroxylase cells impairs chromaffin cell survival.

Glucocorticoid hormones (GCs) have been thought to determine the fate of chromaffin cells from sympathoadrenal progenitor cells. The analysis of mice carrying a germ line deletion of the glucocorticoid receptor (GR) gene has challenged these previous results because the embryonic development of adrenal chromaffin cells is largely unaltered. In the present study, we have analyzed the role of GC-dependent signaling in the postnatal development of adrenal chromaffin cells by conditional inactivation of the GR gene in cells expressing dopamine-beta-hydroxylase, an enzyme required for the synthesis of noradrenaline and adrenaline. These mutant mice are viable, allowing to study whether in the absence of GC signaling further development of the adrenal medulla is affected. Our analysis shows that the loss of GR leads not only to the loss of phenylethanolamine-N-methyl-transferase expression and, therefore, to inhibition of adrenaline synthesis, but also to a dramatic reduction in the number of adrenal chromaffin cells. We provide evidence that increased apoptotic cell death is the main consequence of GR loss. These findings define the essential role of GCs for survival of chromaffin cells and underscore the specific requirement of GCs for adrenergic chromaffin cell differentiation and maintenance.

Vascular normalization in Rgs5-deficient tumours promotes immune destruction.

The vasculature of solid tumours is morphologically aberrant and characterized by dilated and fragile vessels, intensive vessel sprouting and loss of hierarchical architecture. Constant vessel remodelling leads to spontaneous haemorrhages and increased interstitial fluid pressure in the tumour environment. Tumour-related angiogenesis supports tumour growth and is also a major obstacle for successful immune therapy as it prevents migration of immune effector cells into established tumour parenchyma. The molecular mechanisms for these angiogenic alterations are largely unknown. Here we identify regulator of G-protein signalling 5 (Rgs5) as a master gene responsible for the abnormal tumour vascular morphology in mice. Loss of Rgs5 results in pericyte maturation, vascular normalization and consequent marked reductions in tumour hypoxia and vessel leakiness. These vascular and intratumoral changes enhance influx of immune effector cells into tumour parenchyma and markedly prolong survival of tumour-bearing mice. This is the first demonstration, to our knowledge, of reduced tumour angiogenesis and improved immune therapeutic outcome on loss of a vascular gene function and establishes a previously unrecognized role of G-protein signalling in tumour angiogenesis.

Iron regulatory proteins are essential for intestinal function and control key iron absorption molecules in the duodenum.

Iron regulatory proteins (IRPs) orchestrate the posttranscriptional regulation of critical iron metabolism proteins at the cellular level. Redundancy between IRP1 and IRP2 associated with embryonic lethality of doubly IRP-deficient mice has precluded the study of IRP function in vivo. Here we use Cre/Lox technology to generate viable organisms lacking IRP expression in a single tissue, the intestine. Mice lacking intestinal IRP expression develop intestinal malabsorption and dehydration postnatally and die within 4 weeks of birth. We demonstrate that IRPs control the expression of divalent metal transporter 1 (DMT1) mRNA and protein, a limiting intestinal iron importer. IRPs are also shown to be critically important to secure physiological levels of the basolateral iron exporter ferroportin. IRPs are thus essential for intestinal function and organismal survival and coordinate the synthesis of key iron metabolism proteins in the duodenum.

Sulfated glycosphingolipid as mediator of phagocytosis: SM4s enhances apoptotic cell clearance and modulates macrophage activity.

Sulfoglycolipids are present on the surface of a variety of cells. The sulfatide SM4s is increased in lung, renal, and colon cancer and is associated with an adverse prognosis, possibly due to a low immunoreactivity of the tumor. As macrophages significantly contribute to the inflammatory infiltrate in malignancies, we postulated that SM4s may modulate macrophage function. We have investigated the effect of SM4s on the uptake of apoptotic tumor cells, macrophage cytokine profile, and receptor expression. Using flow cytometry and microscopic analyses, we found that coating apoptotic murine carcinoma cells from the colon and kidney with SM4s promoted their phagocytosis by murine macrophages up to 3-fold ex vivo and in vivo. This increased capacity was specifically inhibited by preincubation of macrophages with oxidized or acetylated low density lipoprotein and maleylated albumin, indicating involvement of scavenger receptors in this interaction. The uptake of SM4s-coated apoptotic cells significantly enhanced macrophage production of TGF-beta1, expression of P-selectin, and secretion of IL-6. These data suggest that SM4s within tumors may promote apoptotic cell removal and alter the phenotype of tumor-associated macrophages.

Integrity and barrier function of the epidermis critically depend on glucosylceramide synthesis.

Ceramides are vital components of the water barrier in mammalian skin. Epidermis-specific, a major ceramide portion contains omega-hydroxy very long chain fatty acids (C30-C36). These omega-hydroxy ceramides (Cers) are found in the extracellular lamellae of the stratum corneum either as linoleic acyl esters or protein bound. Glucosylceramide is the major glycosphingolipid of the epidermis. Synthesized from ceramide and UDP-glucose, it is thought to be itself an intracellular precursor and carrier for extracellular omega-hydroxy ceramides. To investigate whether GlcCer is an obligatory intermediate in ceramide metabolism to maintain epidermal barrier function, a mouse with an epidermis-specific glucosylceramide synthase (Ugcg) deficiency has been generated. Four days after birth animals devoid of GlcCer synthesis in keratinocytes showed a pronounced desquamation of the stratum corneum and extreme transepidermal water loss leading to death. The stratum corneum appeared as a thick unstructured mass. Lamellar bodies of the stratum granulosum did not display the usual ordered inner structure and were often irregularly arranged. Although the total amount of epidermal protein-bound ceramides remained unchanged, epidermal-free omega-hydroxy ceramides increased 4-fold and omega-hydroxy sphingomyelins, almost not detectable in wild type epidermis, emerged in quantities comparable with lost GlcCer. We conclude that the transient formation of GlcCer is vital for a regular arrangement of lipids and proteins in lamellar bodies and for the maintenance of the epidermal barrier.