Membrane-associated Molecules Research Articles

Toward the understanding of biology of oocyte life cycle in Xenopus Laevis: No oocytes left behind.

BackgroundFor the past more than 25 years, we have been focusing on the developmental and reproductive biology of the female gametes, oocytes, and eggs, of the African clawed frog Xenopus laevis.MethodsThe events associated with the life cycle of these cells can be classified into the four main categories: first, oogenesis and cell growth in the ovary during the first meiotic arrest; second, maturation and ovulation that occur simultaneously and result in the acquisition of fertilization competence and the second meiotic arrest; third, fertilization, that is sperm‐induced transition from egg to zygote; and fourth, egg death after spontaneous activation in the absence of fertilizing sperm.Main findingsOur studies have demonstrated that signal transduction system involving tyrosine kinase Src and other oocyte/egg membrane‐associated molecules such as uroplakin III and some other cytoplasmic proteins such as mitogen‐activated protein kinase (MAPK) play important roles for successful ovulation, maturation, fertilization, and initiation of embryonic development.ConclusionWe summarize recent advances in understanding cellular and molecular mechanisms underlying life cycle events of the oocytes and eggs. Our further intention is to discuss and predict potentially promising impact of the recent findings on the challenges facing reproductive biology and medicine, as well as societal contexts.

Cold Atmospheric Plasma and Plasma-Activated Medium Trigger RONS-Based Tumor Cell Apoptosis

The selective in vitro anti-tumor mechanisms of cold atmospheric plasma (CAP) and plasma-activated media (PAM) follow a sequential multi-step process. The first step involves the formation of primary singlet oxygen (1O2) through the complex interaction between NO2− and H2O2.1O2 then inactivates some membrane-associated catalase molecules on at least a few tumor cells. With some molecules of their protective catalase inactivated, these tumor cells allow locally surviving cell-derived, extracellular H2O2 and ONOO─ to form secondary 1O2. These species continue to inactivate catalase on the originally triggered cells and on adjacent cells. At the site of inactivated catalase, cell-generated H2O2 enters the cell via aquaporins, depletes glutathione and thus abrogates the cell’s protection towards lipid peroxidation. Optimal inactivation of catalase then allows efficient apoptosis induction through the HOCl signaling pathway that is finalized by lipid peroxidation. An identical CAP exposure did not result in apoptosis for nonmalignant cells. A key conclusion from these experiments is that tumor cell-generated RONS play the major role in inactivating protective catalase, depleting glutathione and establishing apoptosis-inducing RONS signaling. CAP or PAM exposure only trigger this response by initially inactivating a small percentage of protective membrane associated catalase molecules on tumor cells.

Dynamics of Singlet Oxygen-Triggered, RONS-Based Apoptosis Induction after Treatment of Tumor Cells with Cold Atmospheric Plasma or Plasma-Activated Medium

Treatment of tumor cells with cold atmospheric plasma (CAP) or with plasma-activated medium (PAM) leads to a biochemical imprint on these cells. This imprint is mediated by primary singlet oxygen, which is mainly generated through the interaction between CAP-derived H2O2 and NO2−. This imprint is induced with a low efficiency as local inactivation of a few membrane-associated catalase molecules. As sustained generation of secondary singlet oxygen by the tumor cells is activated at the site of the imprint, a rapid bystander effect-like spreading of secondary singlet oxygen generation and catalase inactivation within the cell population is thus induced. This highly dynamic process is essentially driven by NOX1 and NOS of the tumor cells, and finally leads to intercellular RONS-driven apoptosis induction. This dynamic process can be studied by kinetic analysis, combined with the use of specific inhibitors at defined time intervals. Alternatively, it can be demonstrated and quantified by transfer experiments, where pretreated cells are mixed with untreated cells and bystander signaling is determined. These studies allow to conclude that the specific response of tumor cells to generate secondary singlet oxygen is the essential motor for their self-destruction, after a singlet oxygen-mediated triggering process by CAP or PAM.

Anterior-enriched filopodia create the appearance of asymmetric membrane microdomains in polarizing C. elegans zygotes.

ABSTRACTThe association of molecules within membrane microdomains is critical for the intracellular organization of cells. During polarization of the C. elegans zygote, both polarity proteins and actomyosin regulators associate within dynamic membrane-associated foci. Recently, a novel class of asymmetric membrane-associated structures was described that appeared to be enriched in phosphatidylinositol 4,5-bisphosphate (PIP2), suggesting that PIP2 domains could constitute signaling hubs to promote cell polarization and actin nucleation. Here, we probe the nature of these domains using a variety of membrane- and actin cortex-associated probes. These data demonstrate that these domains are filopodia, which are stimulated transiently during polarity establishment and accumulate in the zygote anterior. The resulting membrane protrusions create local membrane topology that quantitatively accounts for observed local increases in the fluorescence signal of membrane-associated molecules, suggesting molecules are not selectively enriched in these domains relative to bulk membrane and that the PIP2 pool as revealed by PHPLCδ1 simply reflects plasma membrane localization. Given the ubiquity of 3D membrane structures in cells, including filopodia, microvilli and membrane folds, similar caveats are likely to apply to analysis of membrane-associated molecules in a broad range of systems.

Mechanistic study of PpIX accumulation using the JFCR39 cell panel revealed a role for dynamin 2-mediated exocytosis

5-aminolevulinic acid (5-ALA) has recently been employed for photodynamic diagnosis (ALA-PDD) and photodynamic therapy (ALA-PDT) of various types of cancer because hyperproliferating tumor cells do not utilize oxidative phosphorylation and do not efficiently produce heme; instead, they accumulate protoporphyrin IX (PpIX), which is a precursor of heme that is activated by violet light irradiation that results in the production of red fluorescence and singlet oxygen. The efficiencies of ALA-PDD and ALA-PDT depend on the efficient cellular uptake of 5-ALA and the inefficient excretion of PpIX. We employed the JFCR39 cell panel to determine whether tumor cells originating from different tissues can produce and accumulate PpIX. We also investigated cellular factors/molecules involved in PpIX excretion by tumor cells with the JFCR39 cell panel. Unexpectedly, the expression levels of ABCG2, which has been considered to play a major role in PpIX extracellular transport, did not show a strong correlation with PpIX excretion levels in the JFCR39 cell panel, although an ABCG2 inhibitor significantly increased intracellular PpIX accumulation in several tumor cell lines. In contrast, the expression levels of dynamin 2, which is a cell membrane-associated molecule involved in exocytosis, were correlated with the PpIX excretion levels. Moreover, inhibitors of dynamin significantly suppressed PpIX excretion and increased the intracellular levels of PpIX. This is the first report demonstrating the causal relationship between dynamin 2 expression and PpIX excretion in tumor cells.

Trogocytosis-Mediated Intracellular Signaling in CD4+ T Cells Drives TH2-Associated Effector Cytokine Production and Differentiation.

CD4+ T cells have been observed to acquire APC-derived membrane and membrane-associated molecules through trogocytosis in diverse immune settings. Despite this, the consequences of trogocytosis on the recipient T cell remain largely unknown. We previously reported that trogocytosed molecules on CD4+ T cells engage their respective surface receptors, leading to sustained TCR signaling and survival after APC removal. Using peptide-pulsed bone marrow-derived dendritic cells and transfected murine fibroblasts expressing antigenic MHC:peptide complexes as APC, we show that trogocytosis-positive CD4+ T cells display effector cytokines and transcription factor expression consistent with a TH2 phenotype. In vitro-polarized TH2 cells were found to be more efficient at performing trogocytosis than TH1 or nonpolarized CD4+ cells, whereas subsequent trogocytosis-mediated signaling induced TH2 differentiation in polarized TH1 and nonpolarized cells. Trogocytosis-positive CD4+ T cells generated in vivo also display a TH2 phenotype in both TCR-transgenic and wild-type models. These findings suggest that trogocytosis-mediated signaling impacts CD4+ T cell differentiation and effector cytokine production and may play a role in augmenting or shaping a TH2-dominant immune response.

Membrane-associated epithelial cell adhesion molecule is slowly cleaved by γ-secretase prior to efficient proteasomal degradation of its intracellular domain

Regulated intramembrane proteolysis (RIP) is a key mechanism for activating transmembrane proteins such as epithelial cell adhesion molecule (EpCAM) for cellular signaling and degradation. EpCAM is highly expressed in carcinomas and progenitor and embryonic stem cells and is involved in the regulation of cell adhesion, proliferation, and differentiation. Strictly sequential cleavage of EpCAM through RIP involves initial shedding of the extracellular domain by α-secretase (ADAM) and β-secretase (BACE) sheddases, generating a membrane-tethered C-terminal fragment EpCTF. Subsequently, the rate-limiting γ-secretase complex catalyzes intramembrane cleavage of EpCTF, generating an extracellular EpCAM-Aβ-like fragment and an intracellular EpICD fragment involved in nuclear signaling. Here, we have combined biochemical approaches with live-cell imaging of fluorescent protein tags to investigate the kinetics of γ-secretase-mediated intramembrane cleavage of EpCTF. We demonstrate that γ-secretase-mediated proteolysis of exogenously and endogenously expressed EpCTF is a slow process with a 50% protein turnover in cells ranging from 45 min to 5.5 h. The slow cleavage was dictated by γ-secretase activity and not by EpCTF species, as indicated by cross-species swapping experiments. Furthermore, both human and murine EpICDs generated from EpCTF by γ-secretase were degraded efficiently (94-99%) by the proteasome. Hence, proteolytic cleavage of EpCTF is a comparably slow process, and EpICD generation does not appear to be suited for rapidly transducing extracellular cues into nuclear signaling, but appears to provide steady signals that can be further controlled through efficient proteasomal degradation. Our approach provides an unbiased bioassay to investigate proteolytic processing of EpCTF in single living cells.

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy.

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

Vesicular Delivery of the Antifungal Antibiotics of Lysobacter enzymogenes C3.

Lysobacter enzymogenes C3 is a predatory strain of Gram-negative gliding bacteria that produces antifungal antibiotics by the polyketide synthetic pathway. Outer membrane vesicles (OMV) are formed as a stress response and can deliver virulence factors to host cells. The production of OMV by C3 and their role in antifungal activity are reported here. Vesicles in the range of 130 to 150 nm in diameter were discovered in the cell-free supernatants of C3 cultures. These OMV contain molecules characteristic of bacterial outer membranes, such as lipopolysaccharide and phospholipids. In addition, they contain chitinase activity and essentially all of the heat-stable antifungal activity in cell supernatants. We show here that C3 OMV can directly inhibit growth of the yeast Saccharomyces cerevisiae as well as that of the filamentous fungus Fusarium subglutinans The activity is dependent on physical contact between OMV and the cells. Furthermore, fluorescent lipid labeling of C3 OMV demonstrated transfer of the membrane-associated probe to yeast cells, suggesting the existence of a mechanism of delivery for membrane-associated molecules. Mass spectrometric analysis of C3 OMV extracts indicates the presence of molecules with molecular weights identical to some of the previously identified antifungal products of C3. These data together suggest that OMV act as an important remote mobile component of predation by LysobacterIMPORTANCE The data presented here suggest a newly discovered function of outer membrane vesicles (OMV) that are produced from the outer membrane of the bacterial species Lysobacter enzymogenes strain C3. We show that these OMV can be released from the surface of the cells to deliver antibiotics to target fungal organisms as a mechanism of killing or growth inhibition. Understanding the role of OMV in antibiotic delivery can generally lead to improved strategies for dealing with antibiotic-resistant organisms. These results also add to the evidence that some bacterially produced antibiotics can be discovered and purified using methods designed for isolation of nanoscale vesicles. Information on these systems can lead to better identification of active molecules or design of delivery vehicles for these molecules.

Hydrodynamic trapping measures the interaction between membrane-associated molecules

How membrane proteins distribute and behave on the surface of cells depends on the molecules’ chemical potential. However, measuring this potential, and how it varies with protein-to-protein distance, has been challenging. Here, we present a method we call hydrodynamic trapping that can achieve this. Our method uses the focused liquid flow from a micropipette to locally accumulate molecules protruding above a lipid membrane. The chemical potential, as well as information about the dimensions of the studied molecule, are obtained by relating the degree of accumulation to the strength of the trap. We have used this method to study four representative proteins, with different height-to-width ratios and molecular properties; from globular streptavidin, to the rod-like immune cell proteins CD2, CD4 and CD45. The data we obtain illustrates how protein shape, glycosylation and flexibility influence the behaviour of membrane proteins, as well as underlining the general applicability of the method.

Determinations of Orientation of Membrane-Associated Fluorescent Molecules Made Trivially Simple

Determinations of Orientation of Membrane-Associated Fluorescent Molecules Made Trivially Simple

Computing Curvature Sensitivity of Biomolecules in Membranes by Simulated Buckling.

Membrane curvature sensing, where the binding free energies of membrane-associated molecules depend on the local membrane curvature, is a key factor to modulate and maintain the shape and organization of cell membranes. However, the microscopic mechanisms are not well understood, partly due to absence of efficient simulation methods. Here, we describe a method to compute the curvature dependence of the binding free energy of a membrane-associated probe molecule that interacts with a buckled membrane, which has been created by lateral compression of a flat bilayer patch. This buckling approach samples a wide range of curvatures in a single simulation, and anisotropic effects can be extracted from the orientation statistics. We develop an efficient and robust algorithm to extract the motion of the probe along the buckled membrane surface, and evaluate its numerical properties by extensive sampling of three coarse-grained model systems: local lipid density in a curved environment for single-component bilayers, curvature preferences of individual lipids in two-component membranes, and curvature sensing by a homotrimeric transmembrane protein. The method can be used to complement experimental data from curvature partition assays and provides additional insight into mesoscopic theories and molecular mechanisms for curvature sensing.

A polar SxxS motif drives assembly of the transmembrane domains of Toll-like receptor 4

Like all members of the Toll-like receptor (TLR) family, TLR4 comprises of a large ectodomain (ECD) involved in ligand recognition at the cell-surface, and a cytosolic Toll/interleukin-1 receptor (TIR) signalling domain, linked by a lipid membrane-anchored transmembrane (TM) domain (TMD). Binding of immunostimulatory pathogen-associated molecular patterns (PAMPs) such as bacterial lipopolysaccharide (LPS) to myeloid differentiation factor 2 (MD-2) coreceptor-complexed TLR4 leads to its dimerization, resulting in productive juxtaposition of TIR domains and the initiation of pro-inflammatory innate immune responses. Whilst the process of PAMP recognition is relatively well understood, thanks to numerous high-resolution crystallographic structures of ECDs, the mechanism by which such recognition is translated into TMD dimerization and activating conformational changes is less clear. Based on available biophysical and biochemical experimental data, ab initio modelling, and multiscale molecular dynamics (MD) simulations entailing a total of >13μs and >200μs of atomistic and coarse-grained sampling, respectively, we investigate the structural basis for TLR4 TMD dimerization within a biologically relevant lipid membrane environment. A key polar-xx-polar (637SxxS640) motif is shown to drive association of the TLR4 TMDs, and to maintain a flexible interface, which may be disrupted by selected point mutations. Furthermore, MD simulations of various TMD+ECD constructs have been used to investigate the coupling between domains, revealing that flexible linkers abrogate dimerization via aggregation of ECDs at the membrane surface, explaining previous biochemical observations. These results improve our understanding of the assembly and signalling mechanisms of TLR4, and pave the way for rational structure-based development of membrane-associated immunomodulatory molecules.

Aberrant plasma IL-7 and soluble IL-7 receptor levels indicate impaired T-cell response to IL-7 in human tuberculosis.

T-cell proliferation and generation of protective memory during chronic infections depend on Interleukin-7 (IL-7) availability and receptivity. Regulation of IL-7 receptor (IL-7R) expression and signalling are key for IL-7-modulated T-cell functions. Aberrant expression of soluble (s) and membrane-associated (m) IL-7R molecules is associated with development of autoimmunity and immune failure in acquired immune deficiency syndrome (AIDS) patients. Here we investigated the role of IL-7/IL-7R on T-cell immunity in human tuberculosis. We performed two independent case-control studies comparing tuberculosis patients and healthy contacts. This was combined with follow-up examinations for a subgroup of tuberculosis patients under therapy and recovery. Blood plasma and T cells were characterised for IL-7/sIL-7R and mIL-7R expression, respectively. IL-7-dependent T-cell functions were determined by analysing STAT5 phosphorylation, antigen-specific cytokine release and by analysing markers of T-cell exhaustion and inflammation. Tuberculosis patients had lower soluble IL-7R (p < 0.001) and higher IL-7 (p < 0.001) plasma concentrations as compared to healthy contacts. Both markers were largely independent and aberrant expression normalised during therapy and recovery. Furthermore, tuberculosis patients had lower levels of mIL-7R in T cells caused by post-transcriptional mechanisms. Functional in vitro tests indicated diminished IL-7-induced STAT5 phosphorylation and impaired IL-7-promoted cytokine release of Mycobacterium tuberculosis-specific CD4+ T cells from tuberculosis patients. Finally, we determined T-cell exhaustion markers PD-1 and SOCS3 and detected increased SOCS3 expression during therapy. Only moderate correlation of PD-1 and SOCS3 with IL-7 expression was observed. We conclude that diminished soluble IL-7R and increased IL-7 plasma concentrations, as well as decreased membrane-associated IL-7R expression in T cells, reflect impaired T-cell sensitivity to IL-7 in tuberculosis patients. These findings show similarities to pathognomonic features of impaired T-cell functions and immune failure described in AIDS patients.

Focal Adhesion Kinase Expression in Ameloblastoma: A Preliminary Observational Study.

Focal Adhesion Kinase (FAK) is a membrane-associated molecule, which plays a major role in tumourigenesis by promoting cell survival, proliferation, migration, and invasion. Hence, FAK is a well known molecule that has been targeted therapeutically for the treatment of various malignancies. Ameloblastoma (AB) is a locally invasive, destructive and recurrent benign odontogenic lesion. Based on these properties, we have hypothesized that odontogenic epithelium of AB could show increased expression of FAK. To study the expression of FAK in odontogenic epithelium of AB using immunohistochemistry. Thirty four paraffin embedded AB and 17 Dental Follicle (DF) tissues were retrieved from archives. Immunohistochemical staining was performed using FAK antibody on 5 μm thick formalin fixed paraffin embedded sections. The expression of FAK was studied in ameloblast like cells and stellate reticulum-like cells and was graded as negative, weak and strong. The data was analysed using chi-square test and Fisher's-exact test. In AB, both ameloblast like and stellate reticulum like cells showed positive expression of FAK in the cytoplasm in all the cases (34). The epithelial islands from all the DFs (17) were negative for FAK expression. The difference in the expression of FAK in ameloblast like and stellate reticulum like cells was statistically significant (p<0.0001). Statistically significant difference was observed in the expression of FAK in stellate reticulum like cells of AB and odontogenic epithelial islands of DF (p = 0.0017). All the AB cases showed strong expression of FAK in ameloblast like cells and negative expression was seen in all the cases of DF. FAK was strongly expressed in AB as compared to epithelial islands of DF. FAK could be responsible for the neoplastic nature of AB and possibly play a role in tumour invasiveness.

Estrogen Receptors in Cell Membranes: Regulation and Signaling.

Estrogens can stimulate the development, proliferation, migration, and survival of target cells. These biological effects are mediated through their action upon the plasma membrane estrogen receptors (ERs). ERs regulate transcriptional processes by nuclear translocation and binding to specific response elements, which leads to the regulation of gene expression. This effect is termed genomic or nuclear. However, estrogens may exert their biological activity also without direct binding to DNA and independently of gene transcription or protein synthesis. This action is called non-genomic or non-nuclear. Through non-genomic mechanisms, estrogens can modify regulatory cascades such as MAPK, P13K, and tyrosine cascade as well as membrane-associated molecules such as ion channels and G-protein-coupled receptors. The recent studies on the mechanisms of estrogen action provide an evidence that non-genomic and genomic effects converge. An example of such convergence is the potential possibility to modulate gene expression through these two independent pathways. The understanding of the plasma membrane estrogen receptors is crucial for the development of novel drugs and therapeutic protocols targeting specific receptor actions.

Insights into Adenovirus Uncoating from Interactions with Integrins and Mediators of Host Immunity.

Human adenoviruses are large (150 MDa) nonenveloped double-stranded DNA (dsDNA) viruses that cause acute respiratory, gastrointestinal and ocular infections. Despite these disease associations, adenovirus has aided basic and clinical research efforts through studies of its association with cells and as a target of host antiviral responses. This review highlights the knowledge of adenovirus disassembly and nuclear transport gleaned from structural, biophysical and functional analyses of adenovirus interactions with soluble and membrane-associated host molecules.

Molecular insights into a dinoflagellate bloom.

In coastal waters worldwide, an increase in frequency and intensity of algal blooms has been attributed to eutrophication, with further increases predicted because of climate change. Yet, the cellular-level changes that occur in blooming algae remain largely unknown. Comparative metatranscriptomics was used to investigate the underlying molecular mechanisms associated with a dinoflagellate bloom in a eutrophied estuary. Here we show that under bloom conditions, there is increased expression of metabolic pathways indicative of rapidly growing cells, including energy production, carbon metabolism, transporters and synthesis of cellular membrane components. In addition, there is a prominence of highly expressed genes involved in the synthesis of membrane-associated molecules, including those for the production of glycosaminoglycans (GAGs), which may serve roles in nutrient acquisition and/or cell surface adhesion. Biotin and thiamine synthesis genes also increased expression along with several cobalamin biosynthesis-associated genes, suggesting processing of B12 intermediates by dinoflagellates. The patterns in gene expression observed are consistent with bloom-forming dinoflagellates eliciting a cellular response to elevated nutrient demands and to promote interactions with their surrounding bacterial consortia, possibly in an effort to cultivate for enhancement of vitamin and nutrient exchanges and/or direct consumption. Our findings provide potential molecular targets for bloom characterization and management efforts.

Nuclear factor erythroid 2-related factor 2 a master regulator of retinal vascular regeneration

In this issue of PNAS, Wei and colleagues demonstrate that the Nuclear factor erythroid 2-related factor 2 (Nrf2), a well-known cell-intrinsic cytoprotective factor and critical regulator of the anti-oxidant response plays an important function in reparative angiogenesis by suppressing the antiangiogenic effects of Semaphorin 6A (Sema6A), a membrane-associated guidance molecule expressed by neurons during retinal ischemia (1).

Inhibition of Pancreatic Cancer Cell-Induced Paracrine Hedgehog Signaling by Liver X Receptor Agonists and Oxy16, a Naturally Occurring Oxysterol

The widespread involvement of the Hedgehog (Hh) signaling pathway in human malignancies has driven efforts to develop Hh pathway inhibitors as anti-cancer agents. The majority of these agents antagonize Smoothened (Smo), a plasma membrane-associated signal transducer molecule. However, several such Smo antagonists have failed in clinical trials to benefit patients with cancers that arise from aberrant Hh signaling (which often bypasses Smo). In this study, we report that a naturally occurring oxysterol, 20α, 22(R)-dihydroxycholesterol (Oxy16), a known metabolite in the biosynthesis of steroid hormones, strongly inhibits Hh signaling induced in C3H10T1/2 embryonic fibroblasts and NIH3T3-E1 fibroblasts through a mechanism that is independent of liver X receptor (LXR) activation. We demonstrate that Oxy16 inhibits Hh signaling in Suppressor of Fused (Sufu) null mouse embryonic fibroblast (MEF) cells, indicating that its inhibitory effect on Hh signaling is epistatic to Sufu. We further demonstrate that Oxy16 inhibits Gli1 transcriptional activity in NIH3T3-E1 cells overexpressing Gli1 and a Gli-dependent reporter construct. Altogether, data presented here suggest that Oxy16 may be a suitable starting point for the development of new drugs that inhibit Hh signaling downstream of Smo. By targeting aberrant Hh signaling, such novel Hh pathway inhibitors could significantly broaden the range of clinical applications compared to existing Smo antagonists. Furthermore, the present study adds a new facet to the spectrum of Hh pathway modulation that naturally occurring oxysterol derivatives are capable of, ranging from allosteric activation of the pathway via Smo binding to inhibition of the pathway downstream of Smo. J. Cell. Biochem. 118: 499-509, 2017. © 2016 Wiley Periodicals, Inc.