Oliver Wessely

Prooncogenic Factors miR-23b and miR-27b Are Regulated by Her2/Neu, EGF, and TNF-a in Breast Cancer

miRNAs (miR) are a critical class of small (21-25 nucleotides) noncoding endogenous RNAs implicated in gene expression regulation. We identified miR-23b and miR-27b as miRNAs that are highly upregulated in human breast cancer. We found that engineered knockdown of miR-23b and miR-27b substantially repressed breast cancer growth. Nischarin (NISCH) expression was augmented by knockdown of miR-23b as well as miR-27b. Notably, these miRNAs and Nischarin were inversely expressed in human breast cancers, underscoring their biologic relevance. We showed the clinical relevance of the expression of these miRNAs and showed that high expression of miR-23b and miR-27b correlates with poor outcome in breast cancer. Moreover, intraperitoneally delivered anti-miR-27b restored Nischarin expression and decreased tumor burden in a mouse xenograft model of human mammary tumor. Also, we report for the first time that HER2/neu (ERBB2), EGF, and TNF-α promote miR-23b/27b expression through the AKT/NF-κB signaling cascade. Nischarin was found to regulate miR-27b/23b expression through a feedback loop mechanism by suppressing NF-κB phosphorylation. Because anti-miR-27b compounds that suppress miR-27b inhibit tumor growth, the anti-miR-27b seems to be a good candidate for the development of new antitumor therapies.

Hyperphosphorylation of polycystin-2 at a critical residue in disease reveals an essential role for polycystin-1-regulated dephosphorylation

Mutations in PKD1 (85%) or PKD2 (15%) account for almost all cases of autosomal dominant polycystic kidney disease (ADPKD). The ADPKD proteins, termed as polycystin-1 (PC1) and polycystin-2 (PC2), interact via their C-termini to form a receptor-ion channel complex whose function and regulation are not fully understood. Here, we report the first phosphorylated residue (Ser(829)) in PC2, whose dephosphorylation is mediated by PC1 binding through the recruitment of protein phosphatase-1 alpha (PP1α). Using a new phosphospecific antibody (pPC2) to this site, we demonstrate that Ser(829) is phosphorylated by Protein kinase A (PKA) but remains constitutively phosphorylated in cells and tissues lacking PC1. cAMP increased pSer(829) basolateral localization in MDCK cells in a time dependent manner and was essential for pronephric development in Xenopus embryos. When constitutively expressed, a complex phenotype associated with enhanced ATP-dependent ER Ca(2+) release and loss of growth suppression was observed in cycling cells. These results reveal a reciprocal functional link between PC1 and PC2 which is critically dependent on their interaction. Unopposed cAMP stimulated hyperphosphorylation of PC2 in the absence of functional PC1 could contribute to cyst initiation in PKD1 patients and represents a new molecular paradigm in understanding ADPKD pathogenesis.

Syndecan 4 interacts genetically with Vangl2 to regulate neural tube closure and planar cell polarity

Syndecan 4 (Sdc4) is a cell-surface heparan sulfate proteoglycan (HSPG) that regulates gastrulation, neural tube closure and directed neural crest migration in Xenopus development. To determine whether Sdc4 participates in Wnt/PCP signaling during mouse development, we evaluated a possible interaction between a null mutation of Sdc4 and the loop-tail allele of Vangl2. Sdc4 is expressed in multiple tissues, but particularly in the non-neural ectoderm, hindgut and otic vesicles. Sdc4;Vangl2Lp compound mutant mice have defective spinal neural tube closure, disrupted orientation of the stereocilia bundles in the cochlea and delayed wound healing, demonstrating a strong genetic interaction. In Xenopus, co-injection of suboptimal amounts of Sdc4 and Vangl2 morpholinos resulted in a significantly greater proportion of embryos with defective neural tube closure than each individual morpholino alone. To probe the mechanism of this interaction, we overexpressed or knocked down Vangl2 function in HEK293 cells. The Sdc4 and Vangl2 proteins colocalize, and Vangl2, particularly the Vangl2Lp mutant form, diminishes Sdc4 protein levels. Conversely, Vangl2 knockdown enhances Sdc4 protein levels. Overall HSPG steady-state levels were regulated by Vangl2, suggesting a molecular mechanism for the genetic interaction in which Vangl2Lp/+ enhances the Sdc4-null phenotype. This could be mediated via heparan sulfate residues, as Vangl2Lp/+ embryos fail to initiate neural tube closure and develop craniorachischisis (usually seen only in Vangl2Lp/Lp) when cultured in the presence of chlorate, a sulfation inhibitor. These results demonstrate that Sdc4 can participate in the Wnt/PCP pathway, unveiling its importance during neural tube closure in mammalian embryos.

Expression of Wnt Signaling Components during Xenopus Pronephros Development

Background The formation of the vertebrate kidney is tightly regulated and relies on multiple evolutionarily conserved inductive events. These are present in the complex metanephric kidney of higher vertebrates, but also in the more primitive pronephric kidney functional in the larval stages of amphibians and fish. Wnts have long been viewed as central in this process. Canonical β-Catenin-dependent Wnt signaling establishes kidney progenitors and non-canonical β-Catenin-independent Wnt signaling participate in the morphogenetic processes that form the highly sophisticated nephron structure. While some individual Wnt signaling components have been studied extensively in the kidney, the overall pathway has not yet been analyzed in depth. Methodology/Principal Findings Here we report a detailed expression analysis of all Wnt ligands, receptors and several downstream Wnt effectors during pronephros development in Xenopus laevis using in situ hybridization. Out of 19 Wnt ligands, only three, Wnt4, Wnt9a and Wnt11, are specifically expressed in the pronephros. Others such as Wnt8a are present, but in a broader domain comprising adjacent tissues in addition to the kidney. The same paradigm is observed for the Wnt receptors and its downstream signaling components. Fzd1, Fzd4, Fzd6, Fzd7, Fzd8 as well as Celsr1 and Prickle1 show distinct expression domains in the pronephric kidney, whereas the non-traditional Wnt receptors, Ror2 and Ryk, as well as the majority of the effector molecules are rather ubiquitous. In addition to this spatial regulation, the timing of expression is also tightly regulated. In particular, non-canonical Wnt signaling seems to be restricted to later stages of pronephros development. Conclusion/Significance Together these data suggest a complex cross talk between canonical and non-canonical Wnt signaling is required to establish a functional pronephric kidney.

A Comparative Analysis of Glomerulus Development in the Pronephros of Medaka and Zebrafish

The glomerulus of the vertebrate kidney links the vasculature to the excretory system and produces the primary urine. It is a component of every single nephron in the complex mammalian metanephros and also in the primitive pronephros of fish and amphibian larvae. This systematic work highlights the benefits of using teleost models to understand the pronephric glomerulus development. The morphological processes forming the pronephric glomerulus are astoundingly different between medaka and zebrafish. (1) The glomerular primordium of medaka - unlike the one of zebrafish - exhibits a C-shaped epithelial layer. (2) The C-shaped primordium contains a characteristic balloon-like capillary, which is subsequently divided into several smaller capillaries. (3) In zebrafish, the bilateral pair of pronephric glomeruli is fused at the midline to form a glomerulus, while in medaka the two parts remain unmerged due to the interposition of the interglomerular mesangium. (4) Throughout pronephric development the interglomerular mesangial cells exhibit numerous cytoplasmic granules, which are reminiscent of renin-producing (juxtaglomerular) cells in the mammalian afferent arterioles. Our systematic analysis of medaka and zebrafish demonstrates that in fish, the morphogenesis of the pronephric glomerulus is not stereotypical. These differences need be taken into account in future analyses of medaka mutants with glomerulus defects.

Re)Building a Kidney

(Re)Building a Kidney is a National Institute of Diabetes and Digestive and Kidney Diseases-led consortium to optimize approaches for the isolation, expansion, and differentiation of appropriate kidney cell types and the integration of these cells into complex structures that replicate human kidney function. The ultimate goals of the consortium are two-fold: to develop and implement strategies for in vitro engineering of replacement kidney tissue, and to devise strategies to stimulate regeneration of nephrons in situ to restore failing kidney function. Projects within the consortium will answer fundamental questions regarding human gene expression in the developing kidney, essential signaling crosstalk between distinct cell types of the developing kidney, how to derive the many cell types of the kidney through directed differentiation of human pluripotent stem cells, which bioengineering or scaffolding strategies have the most potential for kidney tissue formation, and basic parameters of the regenerative response to injury. As these projects progress, the consortium will incorporate systematic investigations in physiologic function of in vitro and in vivo differentiated kidney tissue, strategies for engraftment in experimental animals, and development of therapeutic approaches to activate innate reparative responses.

MicroRNAs are critical regulators of tuberous sclerosis complex and mTORC1 activity in the size control of the Xenopus kidney

Significance The kidney is an essential organ to remove metabolic waste products and retain essential nutrients. To successfully execute these processes, the kidney and its functional unit, the nephron, must be correctly proportioned. Surprisingly, little is known about the processes governing kidney size. Here, we use the amphibian pronephric kidney as a paradigm to study the molecular mechanisms regulating size control. We show for the first time, to our knowledge, that growth factors belonging to the Insulin growth factor family are critically important in establishing kidney size. Moreover, we demonstrate that the strength of the signal is dependent on the presence of small noncoding RNAs termed microRNAs. These provide robustness to size control and ascertain that the kidney develops properly. MicroRNAs (miRNAs) are major posttranscriptional regulators of a wide variety of biological processes. However, redundancy among most miRNAs has made it difficult to identify their in vivo functions. We previously demonstrated that global inhibition of miRNA biogenesis in Xenopus resulted in a dramatically smaller pronephric kidney. This suggested that microRNAs play a pivotal role in organ size control. Here we now provide a detailed mechanistic explanation for this phenotype. We identified that the activation of the mechanistic target of rapamycin complex 1 (mTORC1) by Insulin and insulin-like growth factor (Igf) 2 is an important regulator in kidney growth, which in turn is modulated by microRNAs. Molecular analyses demonstrate that microRNAs set a threshold for mTORC1 signaling by down-regulating one of its core negative regulators, tuberous sclerosis 1 (Tsc1). Most importantly, this rheostat can be reprogrammed experimentally. Whereas knockdown of miRNAs causes growth arrest, concomitant knockdown of Tsc1 restores mTORC1 activity and proximal tubular size. Together, these data establish a previously unidentified in vivo paradigm for the importance of posttranscriptional regulation in organ size control.

The Polycystin-1, Lipoxygenase, and α-Toxin Domain Regulates Polycystin-1 Trafficking

Mutations in polycystin-1 (PC1) give rise to autosomal dominant polycystic kidney disease, an important and common cause of kidney failure. Despite its medical importance, the function of PC1 remains poorly understood. Here, we investigated the role of the intracellular polycystin-1, lipoxygenase, and α-toxin (PLAT) signature domain of PC1 using nuclear magnetic resonance, biochemical, cellular, and in vivo functional approaches. We found that the PLAT domain targets PC1 to the plasma membrane in polarized epithelial cells by a mechanism involving the selective binding of the PLAT domain to phosphatidylserine and L-α-phosphatidylinositol-4-phosphate (PI4P) enriched in the plasma membrane. This process is regulated by protein kinase A phosphorylation of the PLAT domain, which reduces PI4P binding and recruits β-arrestins and the clathrin adaptor AP2 to trigger PC1 internalization. Our results reveal a physiological role for the PC1-PLAT domain in renal epithelial cells and suggest that phosphorylation-dependent internalization of PC1 is closely linked to its function in renal development and homeostasis.

Protein composition and movements of membrane swellings associated with primary cilia.

Dysfunction of many ciliary proteins has been linked to a list of diseases, from cystic kidney to obesity and from hypertension to mental retardation. We previously proposed that primary cilia are unique communication organelles that function as microsensory compartments that house mechanosensory molecules. Here we report that primary cilia exhibit membrane swellingsxa0or ciliary bulbs, which based on their unique ultrastructure and motility, could be mechanically regulated by fluid-shear stress. Together with the ultrastructure analysis of the swelling, which contains monosialodihexosylganglioside (GM3), our results show that ciliaryxa0bulb has a distinctive set of functional proteins, including GM3 synthase (GM3S), bicaudal-c1 (Bicc1), and polycystin-2 (PC2). In fact, results from our cilia isolation demonstrated for the first time that GM3S and Bicc1 are members of the primary cilia proteins. Although these proteins are not required for ciliary membrane swelling formation under static condition, fluid-shear stress induced swelling formation is partially modulated by GM3S. We therefore propose that the ciliaryxa0bulb exhibits a sensory function within the mechano-ciliary structure. Overall, our studies provided an important step towards understanding the ciliaryxa0bulb function and structure.

Sterol carrier protein 2 regulates proximal tubule size in the Xenopus pronephric kidney by modulating lipid rafts

The kidney is a homeostatic organ required for waste excretion and reabsorption of water, salts and other macromolecules. To this end, a complex series of developmental steps ensures the formation of a correctly patterned and properly proportioned organ. While previous studies have mainly focused on the individual signaling pathways, the formation of higher order receptor complexes in lipid rafts is an equally important aspect. These membrane platforms are characterized by differences in local lipid and protein compositions. Indeed, the cells in the Xenopus pronephric kidney were positive for the lipid raft markers ganglioside GM1 and Caveolin-1. To specifically interfere with lipid raft function in vivo, we focused on the Sterol Carrier Protein 2 (scp2), a multifunctional protein that is an important player in remodeling lipid raft composition. In Xenopus, scp2 mRNA was strongly expressed in differentiated epithelial structures of the pronephric kidney. Knockdown of scp2 did not interfere with the patterning of the kidney along its proximo-distal axis, but dramatically decreased the size of the kidney, in particular the proximal tubules. This phenotype was accompanied by a reduction of lipid rafts, but was independent of the peroxisomal or transcriptional activities of scp2. Finally, disrupting lipid micro-domains by inhibiting cholesterol synthesis using Mevinolin phenocopied the defects seen in scp2 morphants. Together these data underscore the importance for localized signaling platforms in the proper formation of the Xenopus kidney.