AKT signaling promotes DNA damage accumulation and proliferation in polycystic kidney disease.

Polycystic kidney disease (PKD) results in the formation of renal cysts that can impair function leading to renal failure. DNA damage accumulates in renal epithelial cells in PKD, but the molecular mechanisms are unclear and are investigated here. Phosphoinositide 3-kinase (PI3K)/AKT signaling activates mammalian target of rapamycin complex 1 (mTORC1) and hyperactivation of mTORC1 is a common event in PKD; however, mTORC1 inhibitors have yielded disappointing results in clinical trials. Here, we demonstrate AKT and mTORC1 hyperactivation in two representative murine PKD models (renal epithelial-specific Inpp5e knockout and collecting duct-specific Pkd1 deletion) and identify a downstream signaling network that contributes to DNA damage accumulation. Inpp5e- and Pkd1-null renal epithelial cells showed DNA damage including double-stranded DNA breaks associated with increased replication fork numbers, multinucleation and centrosome amplification. mTORC1 activated CAD, which promotes de novo pyrimidine synthesis, to sustain cell proliferation. AKT, but not mTORC1, inhibited the DNA repair/replication fork origin firing regulator TOPBP1, which impacts on DNA damage and cell proliferation. Notably, Inpp5e- and Pkd1-null renal epithelial cell spheroid formation defects were rescued by AKT inhibition. These data reveal that AKT hyperactivation contributes to DNA damage accumulation in multiple forms of PKD and cooperates with mTORC1 to promote cell proliferation. Hyperactivation of AKT may play a causal role in PKD by regulating DNA damage and cell proliferation, independent of mTORC1, and AKT inhibition may be a novel therapeutic approach for PKD.

Anti-tumor Effect of Folate-Binding Protein: In Vitro and In Vivo Studies.

Folate receptor (FR) overexpression in a wide range of solid tumors provides an opportunity to develop novel, targeted cancer therapeutics. In this study, we investigated whether prebinding the chemotherapeutic methotrexate (MTX) to folate-binding protein (FBP), the soluble form of FR, would enable the protein to serve as a targeted therapeutic vector, enhancing uptake into tumor cells and improving therapeutic efficacy. In an in vivo study, using an FR-overexpressing KB xenograft model in SCID mice, modest improvement in inhibiting tumor growth was observed for the MTX/FBP mixtures as compared to saline control and free MTX. Surprisingly, FBP alone inhibited tumor growth compared to saline control, free MTX, and FBP/MTX. In order to better understand this effect, we investigated the cytotoxicity of micromolar concentrations of FBP in vitro using the KB, HeLa, and A549 cancer cell lines. Our results revealed concentration-dependent apoptosis (24 h; 10-50 µM) in all three cell lines accompanied by a time- and concentration-dependent reduction (6, 12, and 24 h; 10-50 µM) in metabolic activity and compromised cell plasma membrane integrity. This study demonstrates an apoptosis pathway for cytotoxicity of FBP, an endogenous serum protein, in cancer cell lines with widely varying levels of FR expression. Furthermore, in vivo tumor growth suppression for xenograft KB tumors in SCID mice was observed. These studies suggest novel strategies for the elimination of cancer cells employing endogenous, serum transport proteins.

ß-catenin ablation exacerbates polycystic kidney disease progression.

Polycystic kidney disease (PKD) results from excessive renal epithelial cell proliferation, leading to the formation of large fluid filled cysts which impair renal function and frequently lead to renal failure. Hyperactivation of numerous signaling pathways is hypothesized to promote renal epithelial cell hyperproliferation including mTORC1, extracellular signal-regulated kinase (ERK) and WNT signaling. ß-catenin and its target genes are overexpressed in some PKD models and expression of activated ß-catenin induces cysts in mice; however, ß-catenin murine knockout studies indicate it may also inhibit cystogenesis. Therefore, it remains unclear whether ß-catenin is pro- or anti-cystogenic and whether its role is canonical WNT signaling-dependent. Here, we investigate whether ß-catenin deletion in a PKD model with hyperactived ß-catenin signaling affects disease progression to address whether increased ß-catenin drives PKD. We used renal epithelial cell specific Inpp5e-null PKD mice which we report exhibit increased ß-catenin and target gene expression in the cystic kidneys. Surprisingly, co-deletion of ß-catenin with Inpp5e in renal epithelial cells exacerbated polycystic kidney disease and renal failure compared to Inpp5e deletion alone, but did not normalize ß-catenin target gene expression. ß-catenin/Inpp5e double-knockout kidneys exhibited increased cyst initiation, cell proliferation and MEK/ERK signaling compared to Inpp5e-null, associated with increased fibrosis, which may collectively contribute to accelerated disease. Therefore, increased ß-catenin and WNT target gene expression are not necessarily cyst promoting. Rather ß-catenin may play a dual and context-dependent role in PKD and in the presence of other cyst-inducing mutations (Inpp5e-deletion); ß-catenin loss may exacerbate disease in a WNT target gene-independent manner.

The myotubularin MTMR4 regulates phagosomal phosphatidylinositol 3-phosphate turnover and phagocytosis.

Macrophage phagocytosis is required for effective clearance of invading bacteria and other microbes. Coordinated phosphoinositide signaling is critical both for phagocytic particle engulfment and subsequent phagosomal maturation to a degradative organelle. Phosphatidylinositol 3-phosphate (PtdIns(3)P) is a phosphoinositide that is rapidly synthesized and degraded on phagosomal membranes, where it recruits FYVE domain- and PX motif-containing proteins that promote phagosomal maturation. However, the molecular mechanisms that regulate PtdIns(3)P removal from the phagosome have remained unclear. We report here that a myotubularin PtdIns(3)P 3-phosphatase, myotubularin-related protein-4 (MTMR4), regulates macrophage phagocytosis. MTMR4 overexpression reduced and siRNA-mediated Mtmr4 silencing increased levels of cell-surface immunoglobulin receptors (i.e. Fcγ receptors (FcγRs)) on RAW 264.7 macrophages, associated with altered pseudopodal F-actin. Furthermore, MTMR4 negatively regulated the phagocytosis of IgG-opsonized particles, indicating that MTMR4 inhibits FcγR-mediated phagocytosis, and was dynamically recruited to phagosomes of macrophages during phagocytosis. MTMR4 overexpression decreased and Mtmr4-specific siRNA expression increased the duration of PtdIns(3)P on phagosomal membranes. Macrophages treated with Mtmr4-specific siRNA were more resistant to Mycobacterium marinum-induced phagosome arrest, associated with increased maturation of mycobacterial phagosomes, indicating that extended PtdIns(3)P signaling on phagosomes in the Mtmr4-knockdown cells permitted trafficking of phagosomes to acidic late endosomal and lysosomal compartments. In conclusion, our findings indicate that MTMR4 regulates PtdIns(3)P degradation in macrophages and thereby controls phagocytosis and phagosomal maturation.

Inpp5e suppresses polycystic kidney disease via inhibition of PI3K/Akt-dependent mTORC1 signaling.

Polycystic kidney disease (PKD) is a common cause of renal failure with few effective treatments. INPP5E is an inositol polyphosphate 5-phosphatase that dephosphorylates phosphoinositide 3-kinase (PI3K)-generated PI(3,4,5)P3 and is mutated in ciliopathy syndromes. Germline Inpp5e deletion is embryonically lethal, attributed to cilia stability defects, and is associated with polycystic kidneys. However, the molecular mechanisms responsible for PKD development upon Inpp5e loss remain unknown. Here, we show conditional inactivation of Inpp5e in mouse kidney epithelium results in severe PKD and renal failure, associated with a partial reduction in cilia number and hyperactivation of PI3K/Akt and downstream mammalian target of rapamycin complex 1 (mTORC1) signaling. Treatment with an mTORC1 inhibitor improved kidney morphology and function, but did not affect cilia number or length. Therefore, we identify Inpp5e as an essential inhibitor of the PI3K/Akt/mTORC1 signaling axis in renal epithelial cells, and demonstrate a critical role for Inpp5e-dependent mTORC1 regulation in PKD suppression.

INPP5E interacts with AURKA, linking phosphoinositide signaling to primary cilium stability.

Mutations in inositol polyphosphate 5-phosphatase E (INPP5E) cause the ciliopathies known as Joubert and MORM syndromes; however, the role of INPP5E in ciliary biology is not well understood. Here, we describe an interaction between INPP5E and AURKA, a centrosomal kinase that regulates mitosis and ciliary disassembly, and we show that this interaction is important for the stability of primary cilia. Furthermore, AURKA phosphorylates INPP5E and thereby increases its 5-phosphatase activity, which in turn promotes transcriptional downregulation of AURKA, partly through an AKT-dependent mechanism. These findings establish the first direct link between AURKA and phosphoinositide signaling and suggest that the function of INPP5E in cilia is at least partly mediated by its interactions with AURKA.

3D-printed surfaces of titanium implant: the fibroblasts response.

Ti-6Al-4V (wt%) is the most widely used titanium alloy and its additive manufactured (or 3D printed) parts with near net-shape have provided great advantages for biomedical applications. While the impact of surface roughness on the biocompatibility of 3D-printed Ti-6Al-4V part is recognized, further exploration is needed to fully understand this complex relationship. Hence, this study presents a comprehensive evaluation of as-printed Ti-6Al-4V structures, both with and without surface texturing, with particular focus on the fibroblast response. Alongside a flat surface, or as-printed surface, two different types of surface textures: diamond texture and diamond crystal texture, were meticulously designed and printed through laser powder bed fusion (LPBF). The viability, cell adhesion, and morphology of human and murine fibroblasts seeded on the surface patterns was investigated, as well as the distribution of extracellular matrix (ECM) proteins (collagen I, fibronectin). The results demonstrated that the as-fabricated surface morphologies did not impact fibroblast viability, however, a reduced density of human fibroblasts was observed on the diamond texture surface, likely owing to the upright strut structure preventing cell adhesion. Interestingly, spreading of the human, but not murine, fibroblasts was limited by the remaining partially-sintered powders. The relative intensity of ECM protein signals was unaffected, however, ECM protein distribution across the surfaces was also altered. Thus, the as-printed substrates, particularly with diamond crystal struts, present a promising avenue for the cost-effective and efficient fabrication of Ti-6Al-4V components for medical applications in the future.

Fallopian tube rheology regulates epithelial cell differentiation and function to enhance cilia formation and coordination.

The rheological properties of the extracellular fluid in the female reproductive tract vary spatiotemporally, however, the effect on the behaviour of epithelial cells that line the tract is unexplored. Here, we reveal that epithelial cells respond to the elevated viscosity of culture media by modulating their development and functionality to enhance cilia formation and coordination. Specifically, ciliation increases by 4-fold and cilia beating frequency decreases by 30% when cells are cultured at 100 mPa·s. Further, cilia manifest a coordinated beating pattern that can facilitate the formation of metachronal waves. At the cellular level, viscous loading activates the TRPV4 channel in the epithelial cells to increase intracellular Ca2+, subsequently decreasing the mitochondrial membrane potential level for ATP production to maintain cell viability and function. Our findings provide additional insights into the role of elevated tubal fluid viscosity in promoting ciliation and coordinating their beating-a potential mechanism to facilitate the transport of egg and embryo, suggesting possible therapeutic opportunities for infertility treatment.

Real-time electro-mechanical profiling of dynamically beating human cardiac organoids by coupling resistive skins with microelectrode arrays.

Cardiac organoids differentiated from induced pluripotent stem cells are emerging as a promising platform for pre-clinical drug screening, assessing cardiotoxicity, and disease modelling. However, it is challenging to simultaneously measure mechanical contractile forces and electrophysiological signals of cardiac organoids in real-time and in-situ with the existing methods. Here, we present a biting-inspired sensory system based on a resistive skin sensor and a microelectrode array. The bite-like contact can be established with a micromanipulator to precisely position the resistive skin sensor on the top of the cardiac organoid while the 3D microneedle electrode array probes from underneath. Such reliable contact is key to achieving simultaneous electro-mechanical measurements. We demonstrate the use of our system for modelling cardiotoxicity with the anti-cancer drug doxorubicin. The electro-mechanical parameters described here elucidate the acute cardiotoxic effects induced by doxorubicin. This integrated electro-mechanical system enables a suite of new diagnostic options for assessing cardiac organoids and tissues.

Deletion of Aurora kinase A prevents the development of polycystic kidney disease in mice.

Aurora Kinase A (AURKA) promotes cell proliferation and is overexpressed in different types of polycystic kidney disease (PKD). To understand AURKA's role in regulating renal cyst development we conditionally deleted the gene in mouse models of Autosomal Dominant PKD (ADPKD) and Joubert Syndrome, caused by Polycystin 1 (Pkd1) and Inositol polyphosphate-5-phosphatase E (Inpp5e) mutations respectively. We show that while Aurka is dispensable for collecting duct development and homeostasis, its deletion prevents cyst formation in both disease models. Cross-comparison of transcriptional changes implicated AKT signaling in cyst prevention and we show that (i) AURKA and AKT physically interact, (ii) AURKA regulates AKT activity in a kinase-independent manner and (iii) inhibition of AKT can reduce disease severity. AKT activation also regulates Aurka expression, creating a feed-forward loop driving renal cystogenesis. We find that the AURKA kinase inhibitor Alisertib stabilises the AURKA protein, agonizing its cystogenic functions. These studies identify AURKA as a master regulator of renal cyst development in different types of PKD, functioning in-part via AKT.

Scube activity is necessary for Hedgehog signal transduction in vivo.

The Hedgehog (HH) signaling pathway is a central regulator of embryonic development, controlling the pattern and proliferation of a wide variety of organs. Previous studies have implicated the secreted protein, Scube2, in HH signal transduction in the zebrafish embryo (Hollway et al., 2006; Kawakami et al., 2005; Woods and Talbot, 2005) although the nature of the molecular function of Scube2 in this process has remained undefined. This analysis has been compounded by the fact that removal of Scube2 activity in the zebrafish embryo leads to only subtle defects in HH signal transduction in vivo (Barresi et al., 2000; Hollway et al., 2006; Ochi and Westerfield, 2007; van Eeden et al., 1996; Wolff et al., 2003). Here we present the discovery of two additional scube genes in zebrafish, scube1 and scube3, and demonstrate their roles in facilitating HH signal transduction. Knocking down the function of all three scube genes simultaneously phenocopies a complete loss of HH signal transduction in the embryo, revealing that Scube signaling is essential for HH signal transduction in vivo. We further define the molecular role of scube2 in HH signaling.

Phosphoinositide phosphatases: just as important as the kinases.

Phosphoinositide phosphatases comprise several large enzyme families with over 35 mammalian enzymes identified to date that degrade many phosphoinositide signals. Growth factor or insulin stimulation activates the phosphoinositide 3-kinase that phosphorylates phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P(2)] to form phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P(3)], which is rapidly dephosphorylated either by PTEN (phosphatase and tensin homologue deleted on chromosome 10) to PtdIns(4,5)P(2), or by the 5-phosphatases (inositol polyphosphate 5-phosphatases), generating PtdIns(3,4)P(2). 5-phosphatases also hydrolyze PtdIns(4,5)P(2) forming PtdIns(4)P. Ten mammalian 5-phosphatases have been identified, which regulate hematopoietic cell proliferation, synaptic vesicle recycling, insulin signaling, and embryonic development. Two 5-phosphatase genes, OCRL and INPP5E are mutated in Lowe and Joubert syndrome respectively. SHIP [SH2 (Src homology 2)-domain inositol phosphatase] 2, and SKIP (skeletal muscle- and kidney-enriched inositol phosphatase) negatively regulate insulin signaling and glucose homeostasis. SHIP2 polymorphisms are associated with a predisposition to insulin resistance. SHIP1 controls hematopoietic cell proliferation and is mutated in some leukemias. The inositol polyphosphate 4-phosphatases, INPP4A and INPP4B degrade PtdIns(3,4)P(2) to PtdIns(3)P and regulate neuroexcitatory cell death, or act as a tumor suppressor in breast cancer respectively. The Sac phosphatases degrade multiple phosphoinositides, such as PtdIns(3)P, PtdIns(4)P, PtdIns(5)P and PtdIns(3,5)P(2) to form PtdIns. Mutation in the Sac phosphatase gene, FIG4, leads to a degenerative neuropathy. Therefore the phosphatases, like the lipid kinases, play major roles in regulating cellular functions and their mutation or altered expression leads to many human diseases.

Identification of a proline-rich inositol polyphosphate 5-phosphatase (PIPP)•collapsin response mediator protein 2 (CRMP2) complex that regulates neurite elongation.

Neuron polarization is essential for the formation of one axon and multiple dendrites, establishing the neuronal circuitry. Phosphoinositide 3-kinase (PI3K) signaling promotes axon selection and elongation. Here we report in hippocampal neurons siRNA knockdown of the proline-rich inositol polyphosphate 5-phosphatase (PIPP), which degrades PI3K-generated PtdIns(3,4,5)P(3), results in multiple hyperelongated axons consistent with a polarization defect. We identify collapsin response mediator protein 2 (CRMP2), which regulates axon selection by promoting WAVE1 delivery via Kinesin-1 motors to the axon growth cone, as a PIPP-interacting protein by Y2H screening, direct binding studies, and coimmunoprecipitation of an endogenous PIPP, CRMP2, and Kinesin-1 complex from brain lysates. The C-terminal growth cone-targeting domain of PIPP facilitates its interaction with CRMP2. PIPP growth cone localization is CRMP2-dependent. PIPP knockdown in PC12 cells promotes neurite elongation, WAVE1 and Kinesin-1 growth cone localization, whereas knockdown of CRMP2 exhibits the opposite phenotype, with shorter neurites and decreased WAVE1/Kinesin-1 at the growth cone. In contrast, CRMP2 overexpression promotes neurite elongation, a phenotype rescued by full-length PIPP, or expression of the CRMP2-binding PIPP domain. Therefore this study identifies PIPP and CRMP2 exert opposing roles in promoting axon selection and neurite elongation and the complex between these proteins serves to regulate the localization of effectors that promote neurite extension.

Silencer of death domains (SODD) inhibits skeletal muscle and kidney enriched inositol 5-phosphatase (SKIP) and regulates phosphoinositide 3-kinase (PI3K)/Akt signaling to the actin cytoskeleton.

Phosphoinositide 3-kinase (PI3K) regulates cell polarity and migration by generating phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P(3)) at the leading edge of migrating cells. The serine-threonine protein kinase Akt binds to PI(3,4,5)P(3), resulting in its activation. Active Akt promotes spatially regulated actin cytoskeletal remodeling and thereby directed cell migration. The inositol polyphosphate 5-phosphatases (5-ptases) degrade PI(3,4,5)P(3) to form PI(3,4)P(2), which leads to diminished Akt activation. Several 5-ptases, including SKIP and SHIP2, inhibit actin cytoskeletal reorganization by opposing PI3K/Akt signaling. In this current study, we identify a molecular co-chaperone termed silencer of death domains (SODD/BAG4) that forms a complex with several 5-ptase family members, including SKIP, SHIP1, and SHIP2. The interaction between SODD and SKIP exerts an inhibitory effect on SKIP PI(3,4,5)P(3) 5-ptase catalytic activity and consequently enhances the recruitment of PI(3,4,5)P(3)-effectors to the plasma membrane. In contrast, SODD(-/-) mouse embryonic fibroblasts exhibit reduced Akt-Ser(473) and -Thr(308) phosphorylation following EGF stimulation, associated with increased SKIP PI(3,4,5)P(3)-5-ptase activity. SODD(-/-) mouse embryonic fibroblasts exhibit decreased EGF-stimulated F-actin stress fibers, lamellipodia, and focal adhesion complexity, a phenotype that is rescued by the expression of constitutively active Akt1. Furthermore, reduced cell migration was observed in SODD(-/-) macrophages, which express the three 5-ptases shown to interact with SODD (SKIP, SHIP1, and SHIP2). Therefore, this study identifies SODD as a novel regulator of PI3K/Akt signaling to the actin cytoskeleton.

The myotubularin phosphatase MTMR4 regulates sorting from early endosomes.

Phosphatidylinositol 3-phosphate [PtdIns(3)P] regulates endocytic trafficking and the sorting of receptors through early endosomes, including the rapid recycling of transferrin (Tfn). However, the phosphoinositide phosphatase that selectively opposes this function is unknown. The myotubularins are a family of eight catalytically active and six inactive enzymes that hydrolyse PtdIns(3)P to form PtdIns. However, the role each myotubularin family member plays in regulating endosomal PtdIns(3)P and thereby endocytic trafficking is not well established. Here, we identify the myotubularin family member MTMR4, which localizes to early endosomes and also to Rab11- and Sec15-positive recycling endosomes. In cells with MTMR4 knockdown, or following expression of the catalytically inactive MTMR4, MTMR4(C407A), the number of PtdIns(3)P-decorated endosomes significantly increased. MTMR4 overexpression delayed the exit of Tfn from early endosomes and its recycling to the plasma membrane. By contrast, expression of MTMR4(C407A), which acts as a dominant-negative construct, significantly accelerated Tfn recycling. However, in MTMR4 knockdown cells Tfn recycling was unchanged, suggesting that other MTMs might also contribute to recycling. MTMR4 regulated the subcellular distribution of Rab11 and, in cells with RNAi-mediated knockdown of MTMR4, Rab11 was directed away from the pericentriolar recycling compartment. The subcellular distribution of VAMP3, a v-SNARE protein that resides in recycling endosomes and endosome-derived transport vesicles, was also regulated by MTMR4. Therefore, MTMR4 localizes at the interface of early and recycling endosomes to regulate trafficking through this pathway.

A soft and ultrasensitive force sensing diaphragm for probing cardiac organoids instantaneously and wirelessly.

Time-lapse mechanical properties of stem cell derived cardiac organoids are important biological cues for understanding contraction dynamics of human heart tissues, cardiovascular functions and diseases. However, it remains difficult to directly, instantaneously and accurately characterize such mechanical properties in real-time and in situ because cardiac organoids are topologically complex, three-dimensional soft tissues suspended in biological media, which creates a mismatch in mechanics and topology with state-of-the-art force sensors that are typically rigid, planar and bulky. Here, we present a soft resistive force-sensing diaphragm based on ultrasensitive resistive nanocracked platinum film, which can be integrated into an all-soft culture well via an oxygen plasma-enabled bonding process. We show that a reliable organoid-diaphragm contact can be established by an 'Atomic Force Microscope-like' engaging process. This allows for instantaneous detection of the organoids' minute contractile forces and beating patterns during electrical stimulation, resuscitation, drug dosing, tissue culture, and disease modelling.

Soft Wearable Healthcare Materials and Devices.

In spite of advances in electronics and internet technologies, current healthcare remains hospital-centred. Disruptive technologies are required to translate state-of-art wearable devices into next-generation patient-centered diagnosis and therapy. In this review, recent advances in the emerging field of soft wearable materials and devices are summarized. A prerequisite for such future healthcare devices is the need of novel materials to be mechanically compliant, electrically conductive, and biologically compatible. It is begun with an overview of the two viable design strategies reported in the literatures, which is followed by description of state-of-the-art wearable healthcare devices for monitoring physical, electrophysiological, chemical, and biological signals. Self-powered wearable bioenergy devices are also covered and sensing systems, as well as feedback-controlled wearable closed-loop biodiagnostic and therapy systems. Finally, it is concluded with an overall summary and future perspective.

Analysis of phosphatidylinositol 3,4,5 trisphosphate 5-phosphatase activity by in vitro and in vivo assays.

Phosphatidylinositol 3,4,5 trisphosphate [PtdIns(3,4,5)P3] is a potent membrane-bound signaling molecule transiently synthesized by phosphoinositide 3-kinase (PI3-kinase) in response to extracellular agonists. PtdIns(3,4,5)P3 signals need to be strictly controlled. PtdIns(3,4,5)P3 recruits and binds effectors that function in oncogenic signaling pathways. PtdIns(3,4,5)P3 activates cell proliferation, growth, and migration as well as regulating insulin signaling. The inositol polyphosphate 5-phosphatase family of enzymes dephosphorylate and thereby modulate PtdIns(3,4,5)P3 levels, attenuating PI3-kinase-dependent signaling. PtdIns(3,4,5)P3 5-phosphatase enzyme activity can be assessed in vitro by analysis of the hydrolysis of radiolabeled or fluorescently labeled PtdIns(3,4,5)P3 and in vivo by visualization of the recruitment and turnover of the PtdIns(3,4,5)P3-specific biosensor GFP-PH/ ARNO or other PtdIns(3,4,5)P3 binding proteins at the plasma membrane.

INPP5E regulates phosphoinositide-dependent cilia transition zone function.

Human ciliopathies, including Joubert syndrome (JBTS), arise from cilia dysfunction. The inositol polyphosphate 5-phosphatase INPP5E localizes to cilia and is mutated in JBTS. Murine Inpp5e ablation is embryonically lethal and recapitulates JBTS, including neural tube defects and polydactyly; however, the underlying defects in cilia signaling and the function of INPP5E at cilia are still emerging. We report Inpp5e-/- embryos exhibit aberrant Hedgehog-dependent patterning with reduced Hedgehog signaling. Using mouse genetics, we show increasing Hedgehog signaling via Smoothened M2 expression rescues some Inpp5e-/- ciliopathy phenotypes and "normalizes" Hedgehog signaling. INPP5E's phosphoinositide substrates PI(4,5)P2 and PI(3,4,5)P3 accumulated at the transition zone (TZ) in Hedgehog-stimulated Inpp5e-/- cells, which was associated with reduced recruitment of TZ scaffolding proteins and reduced Smoothened levels at cilia. Expression of wild-type, but not 5-phosphatase-dead, INPP5E restored TZ molecular organization and Smoothened accumulation at cilia. Therefore, we identify INPP5E as an essential point of convergence between Hedgehog and phosphoinositide signaling at cilia that maintains TZ function and Hedgehog-dependent embryonic development.

The SH2 domain containing inositol polyphosphate 5-phosphatase-2: SHIP2.

Phosphoinositides are membrane-bound signaling molecules that recruit, activate and localize target effectors to intracellular membranes regulating apoptosis, cell proliferation, insulin signaling and membrane trafficking. The SH2 domain containing inositol polyphosphate 5-phosphatase-2 (SHIP2) hydrolyzes phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) generating phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2). Overexpression of SHIP2 inhibits insulin-stimulated phosphoinositide 3-kinase (PI3K) dependent signaling events. Analysis of diabetic human subjects has revealed an association between SHIP2 gene polymorphisms and type 2 diabetes mellitus. Genetic ablation of SHIP2 in mice has generated conflicting results. SHIP2 knockout mice were originally reported to show lethal neonatal hypoglycemia resulting from insulin hypersensitivity, but in addition to inactivating the SHIP2 gene, the Phox2a gene was also inadvertently deleted. Another SHIP2 knockout mouse has now been generated which inactivates the SHIP2 gene but leaves Phox2a intact. These animals show normal insulin and glucose tolerance but are highly resistant to weight gain on high fat diets, exhibiting an obesity-resistant phenotype. Therefore, SHIP2 remains a significant therapeutic target for the treatment of both obesity and type 2 diabetes.