The Hedgehog pathway effector Smoothened exhibits signaling competency in the absence of ciliary accumulation
SUMMARY Misactivation of the seven transmembrane protein Smoothened (Smo) is frequently associated with basal cell carcinoma and medulloblastoma. Cellular exposure to secreted Hedgehog (Hh) protein or oncogenic mutations in Hh pathway components induce Smo accumulation in the primary cilium, an antenna-like organelle with mostly unknown cellular functions. Despite the data supporting an indispensible role of the primary cilium in Smo activation, the mechanistic underpinnings of this dependency remain unclear. Using a novel cell membrane impermeable Smo antagonist (IHR-1) we demonstrate that Smo supplied with a synthetic agonist or activated with oncogenic mutations can signal without ciliary accumulation. Similarly, cells with compromised ciliary Smo trafficking due to loss of the phosphatidylinositol-4-phosphate 3-kinase PI3K-C2\xce\xb1 retain transcriptional response to an exogenously supplied Smo agonist. These observations suggest that assembly of a Smo signaling complex in the primary cilium is not a prerequisite for Hh pathway activation driven by Smo agonists or oncogenic Smo molecules.
INTRODUCTION<!>RESULTS<!>DISCUSSION<!>Cell lines and reagents<!>Chemical screen and reporter-based assays<!>Biochemical assays<!>Immunofluorescence<!>Flow cytometry<!>Statistical analysis<!>Chemical synthesis<!>Caco-2 cell permeability<!>Parallel artificial membrane permeability<!>Mass spectrometry<!>Pik3-C2\xce\xb1 silencing<!>RT-PCR<!>Chemically induced SmoM2 maturation assay<!>Radiolabeled SmoM2 pulse-chase experiment
<p>Small molecules that disrupt the Hh signal transduction pathway are targeted therapeutic agents with proven anti-cancer efficacy (Low and de Sauvage, 2010). The foundation of this strategy is chemical inhibitors of Smo, a seven-transmembrane protein with similarity to G-protein coupled receptors (GPCRs) that controls through a signaling cascade the Gli family of DNA binding proteins. Under homeostatic conditions, the twelve-transmembrane protein Patched (Ptch) restrains Smo activity when Ptch is not directly bound to Hh ligand (Ingham and McMahon, 2001). Given the structural similarity of Ptch to small molecule transporters and its activity dependency on residues essential to the action of such transporters, Ptch likely regulates Smo by gating its access to an endogenous small molecule with Smo modulatory activity (Briscoe and Therond, 2013; Taipale et al., 2002). Misactivation of Smo in ~90% of basal cell carcinoma and ~20% of medulloblastoma most commonly results from either loss-of-function mutations in PTCH1 (Hahn et al., 1996; Johnson et al., 1996), or gain-of-function mutations in Smo (Lam et al., 1999; Xie et al., 1998).</p><p>Two pockets that support small molecule-mediated modulation of activity present in Smo further lend support for the existence of endogenous Smo ligands. One pocket is formed by the seven transmembrane (7TM) bundle and another by the extracellular cysteine rich domain (CRD). Whereas the 7TM bundle is accessible to a number of Smo modulators including the anti-cancer agent Vismodegib and a Smo agonist (SAG) (Wang et al., 2014; Wang et al., 2013), the CRD localized pocket binds oxysterols (Myers et al., 2013; Nachtergaele et al., 2013; Nedelcu et al., 2013; Rana et al., 2013). A model of Smo dependent regulation by Ptch that emerges from these studies is that the 7TM bundle constitutes the primary site of Smo regulation by a substrate of Ptch whereas the CRD pocket constitutes an allosteric site that supports maximal Smo activity.</p><p>Activation of the Hh pathway is associated with the accumulation of Smo in the primary cilium, an enigmatic antenna-like cellular structure found in most cells (Goetz and Anderson, 2010). Efforts to understand the importance of Smo subcellular re-distribution in response to Hh using genetic strategies has been hindered by the multiple roles that the primary cilium plays in Hh response including those directly relating to Gli regulation (Ocbina and Anderson, 2008). For example, mutations in some intraflagellar trafficking proteins that support ciliary integrity also inactivate Gli proteins thus compromising functional analysis of Smo-cilium relationships (Ocbina and Anderson, 2008). In addition, the primary cilium is essential to the proteolytic processing of two of the three Gli protein family members (Gli2 and Gli3) into transcriptional repressors in the absence of Hh signaling (Huangfu et al., 2003; Liu et al., 2005).</p><p>The ability of some Smo agonists and antagonists alike to promote Smo accumulation in the primary cilium suggests that this cellular event is not sufficient for pathway activation (Rohatgi et al., 2009; Wang et al., 2012; Wang et al., 2009). Indeed, these observations support a two-step model of Smo activation – Smo accumulation in the primary cilium and its adoption of an active conformation presumably in the primary cilium. Our understanding of how Smo accumulation in the primary cilium and its activation are coupled remains unclear. From a large chemical library screen intended to expand the number of chemical probes useful for studying Hh signaling and cilia biology, we identified several novel pharmacophores that support Smo inhibition. As part of our in-depth study of the most potent compound identified, IHR-1, we observed that Smo bypasses the need to accumulate in the primary cilium for activation when exogenously provided with an agonist or when it harbors an oncogenic mutation. Using ciliary protein trafficking defective cells, we confirm that Smo ciliary accumulation and its ability to induce Gli activation can be uncoupled with the introduction of a Smo agonist. These observations suggest that the assembly of a Smo signaling complex in the primary cilium is not essential for oncogenic Smo signaling.</p><!><p>A small collection of Hh signaling inhibitors [Inhibitor of Hedgehog Response (IHR) compounds] was identified from screening a diverse synthetic chemical library using a cultured cell based reporter of cell autonomous Hh pathway response (Figure 1A and Figure S1A). Following a battery of counter screens to identify specific Hh pathway inhibitors, we retained several potent compounds that do not inhibit other signal transduction pathways (Figure 1B, see Figure S1A, Figure S1B) or disrupt ciliogenesis (Figure S1C) but do disable Hh-mediated transcriptional induction of endogenous targets (Figures S1D and S1E). The seven most potent inhibitors (IHR-1 to −7) target Smo as measured by their ability to block Smo binding to Bodipy-cyclopamine (BD-cyclopamine), a fluorescently labeled Smo antagonist (Figure 1C, Figures S1F and S1G). For the most potent of these seven compounds, IHR-1 (Figure 1B), we also demonstrated direct Smo targeting based on its ability to: a) block Hh-induced movement of Smo into the primary cilium (Figure 1D), b) disengage Smo-dependent abrogation of Gli2 and Gli3 proteolytic processing (Figure 1E, Figures S2A and S2B), and c) inhibit Gli activity induced by loss of PTCH1 (Figure S2C).</p><p>To more directly define the IHR-1 binding site in Smo, we engineered a fluorescently labeled IHR-1 compound that retains activity against Hh pathway response (IHR-Cy3; Figure 1F and Figures S3A-C). Further establishing the utility of IHR-1 as specific probes for Smo, we observed IHR-1 activity and Smo binding are both influenced by its regiochemistry (Figures S3D and S3E). Similar to the results from studies using BD-cyclopamine, the Smo agonist SAG is able to abolish IHR-Cy3 labeling of Smo expressing cells suggesting that IHR-1 and SAG compete for binding to the same pocket formed by the heptahelical bundle (Figure 1G). Further supporting this conclusion, IHR-Cy3 binding to Smo was not disrupted by 20(S)-hydroxycholesterol [20(S)-OHC], which engages the lipid binding pocket in the Smo CRD (see Figure 1G) (Myers et al., 2013; Nachtergaele et al., 2013; Nedelcu et al., 2013).</p><p>Surprisingly, despite its potent activity against Hh-induced pathway activity and its shared binding pocket with SAG, IHR-1 exhibited a markedly reduced maximal inhibitory response for SAG-induced pathway activation (a maximal 100-fold excess of IHR-1 to SAG was evaluated; Figure 2A). Consistent with our ability to compete away IHR-Cy3 cell labeling with SAG and further suggesting that IHR-1 does not function as a partial antagonist is our ability to achieve maximal transcriptional SAG response in the presence of IHR-1 (Figure S3F). Taken together, these observations reveal a population of Smo that is activated by SAG and likely inaccessible to IHR-1. Indeed, SAG is a cell permeable compound that is able to penetrate the blood brain barrier in animal studies suggesting that SAG may activate an intracellular pool of Smo inaccessible to IHR-1 (Heine et al., 2011).</p><p>Our synthetic strategy for achieving the IHR-Cy3 molecule resulted in a derivative (IHR-NAc) that remarkably exhibits improved maximal inhibitory action against SAG-induced Smo response (Figure 2B). At the same time, IHR-NAc exhibited slightly more potent activity than IHR-1 against Hh induced transcriptional response, and retained its ability to block BD-cyclopamine from binding Smo as compared to IHR-1 (see Figure 2B; Figure S4A). Supporting little change in their ability to attack Smo, IHR-1 and IHR-NAc inhibited activity associated with overexpressed wild-type Smo with equal potency (Figure 2C). A difference in the ability of IHR-1 and IHR-NAc to access an intracellular pool of active Smo could explain the poor maximal inhibitory activity of IHR-1 in the presence of SAG. We directly measured the relative cell permeability of IHR-1 and IHR-NAc using a workhorse assay for assessing chemical cell permeability based upon the ability of a molecule of interest to cross a monolayer of Caco-2 cells that form cell-cell tight junctions (Figure 2D) (Li et al., 2007). Our results from this study revealed that IHR-NAc exhibited superior ability as compared to IHR-1 to traverse a cell monolayer (Figure 2E). As Caco-2 cells are derived from the gut and may also utilize active transport mechanisms for drug absorption, we executed a similar test using another workhorse assay for monitoring cell permeability (parallel artificial membrane permeability or PAMPA; Figures 2F and 2G). Both Caco-2 and PAMPA results provide evidence that breaking the symmetry of IHR-1 by changing a chlorine atom into a hydrogen bond donor/acceptor in IHR-1 markedly improves its cell penetration without compromising its ability to inhibit Smo (Figure 2H). Furthermore, we confirm that SAG is membrane permeable using PAMPA.</p><p>The majority of an oncogenic form of Smo (SmoM2) when overexpressed in cultured cells is localized to the endoplasmic reticulum (ER) likely as a consequence of its compromised ability to fold due to the W535L substitution (Chen et al., 2002a). Cell permeable Smo agonists and antagonists are able to promote SmoM2 exit from the ER likely by facilitating protein folding thereby affording a fairly universal assay for monitoring intracellular Smo interaction with chemicals binding the Smo heptahelical cavity (Chen et al., 2002a). In agreement with our assigned relative cell membrane permeability for these chemicals, IHR-NAc but not IHR-1 induced SmoM2 protein to exit the ER (Figures S4B-D). Our cellular activity and cell membrane permeability data taken together reveals a strong correlation between IHR-1/IHR-NAc cell membrane permeability and their ability to inhibit SAG-induced Smo activation.</p><p>In order to better understand the basis for their different activities against SAG- but not Hh-induced pathway response, we next evaluated the effects of IHR-1 and IHR-NAc on several biochemical markers of Hh signaling. Smo activation induces the ciliary depletion of Gpr161, a seven transmembrane protein that regulates cellular cyclic AMP levels and PKA-dependent Gli processing (Mukhopadhyay et al., 2013). Consistent with the effects of IHR-1 and IHR-NAc on SAG-induced Gli activity, IHR-NAc but not IHR-1 blocked SAG-induced Gpr161 ciliary depletion (Figure 3A). Two additional markers of Hh pathway response – abrogation of Gli processing and Gli interaction with its cytoplasmic inhibitory partner Suppressor of Fused (Sufu) – were further examined to confirm the differences in the ability IHR-NAc and IHR-1 to inhibit SAG-induced pathway response (Figure 3B and Figure S5).</p><p>In addition to Gpr161, several other Hh pathway components exhibit subcellular redistribution upon Hh stimulation including Gli2 accumulation at the tip of the primary cilium and Smo accumulation throughout the organelle (Goetz and Anderson, 2010). Perhaps not unexpectedly given the transcriptional response data presented so far, IHR-NAc but not IHR-1 blocked Gli2 accumulation at the ciliary tip in response to SAG treatment (Figure 3C). At the same time, both compounds were equally proficient at inhibiting Smo from accumulating in the primary cilium in the presence of SAG despite the differences in their ability to influence SAG-induced Gli activation (Figure 3D). The ability of IHR-1 and IHR-NAc to block Smo accumulation in the primary cilium reaffirms our position that both chemicals are able to bind to Smo in the presence of SAG. More importantly, it reveals Smo accumulation in the primary cilium can be uncoupled from several well-established biochemical and cell biological events previously associated with Smo activity within the primary cilium.</p><p>We further investigated this observation using a genetically based approach. Loss of the phosphoinositide-3-kinase, class 2, alpha (PI3K-C2α) protein results in ciliary trafficking defects that affect the ability of Smo to accumulate in the primary cilium in response to Hh protein and SAG (Franco et al., 2014). Whereas absence of Smo accumulation is associated with compromised Hh-dependent response in cells with PI3K-C2α loss (Franco et al., 2014), Smo response to SAG stimulation under these conditions remains untested. Consistent with the previous report, cells harboring a Pik3-c2α shRNA construct exhibited loss of PI3K-C2α protein (Figure 4A) and diminished SAG-induced Smo accumulation in the primary cilium (Figures 4B and 4C). On the other hand, the ability of SAG to promote transcription of the well-validated Hh target gene Ptch1 surprisingly was not compromised in cells harboring the Pik3-c2α shRNA (Figure 4D). We find this genetically based observation to be consistent with our conclusion that Smo can signal from a subcellular compartment outside of the primary cilium when activated with SAG.</p><p>The cancer-associated Smo mutations W535L (also known as the M2 mutation) and L412F localize to different TM domains which form the narrow cavity occupied by Smo modulators such as Vismodigeb and SAG (Figure 5A and 5B) (Wang et al., 2014; Wang et al., 2013). Since IHR-1 represents a novel pharmacophore supporting Smo antagonism and likely binds to the heptahelical cavity, we initiated studies to determine if IHR-1 and IHR-NAc may be useful against the constitutively active SmoM2 and SmoL412F molecules. We compared the effects of IHR-1 and IHR-NAc against SmoM2- and SmoL412F-induced Gli activation at a dose well above their respective IC50s in Hh-induced pathway response (Figure 5C). As expected, the FDA-approved Smo antagonist Vismodegib promoted a substantial loss of Smo-dependent Gli activity. Surprisingly, whereas IHR-NAc was as equally effective as Vismodegib, the presence of IHR-1 had limited impact on SmoM2 or SmoL412F signaling. The different sensitivity of SmoM2 to these compounds was also reflected in differences of Gli3 processing and ciliary Gpr161 levels in SmoM2 expressing cells treated with IHR-1 or IHR-NAc (Figures S6A and S6B). At the same time, whereas IHR-NAc but not IHR-1 compromised oncogenic Smo activity, both compounds blunted SmoM2 and SmoL412F accumulation in the primary cilium (Figure 5D). Thus, our observations suggest that SmoM2 and SmoL412F like SAG-bound Smo are capable of eliciting Gli activity from an intracellular compartment outside of the primary cilium. Finally, we note that a SmoM2 molecule lacking a cilia-localization signal was previously shown to be unable to activate Gli activity although it is unclear if this inactivity is due to altered protein folding as a consequence of Smo mutagenesis, an inability to engage a cytoplasmic signaling component, or the absence of localization to the primary cilium (Bijlsma et al., 2012).</p><!><p>Despite the general acceptance that the primary cilium functions as a nexus for Hh signaling in both normal and cancerous contexts (Berbari et al., 2009; Goetz and Anderson, 2010; Han et al., 2009; Wong et al., 2009), our understanding of how this antenna-like organelle contributes to signaling remains rudimentary. The primary cilium's direct support of Gli activation and proteolytic processing has contributed to the difficulties in understanding the organelle's role in the activity of "upstream" pathway components such as Smo (Huangfu and Anderson, 2005; Humke et al., 2010; Liu et al., 2005). Our study has identified a chemically based strategy for uncoupling Smo accumulation in the primary cilium and Gli activation and proteolytic processing regulation (Figure 6). This should facilitate future studies aimed at dissecting the Smo-dependent signal transduction mechanisms in normal and oncogenic settings by minimizing unwanted perturbations to primary cilium function or Smo sequence that can confound subsequent data assessment. Notably, other Smo probes previously shown to exhibit weakened activity for SAG as compared to for Hh-induced pathway response may reflect similar differences in cell membrane permeability (Chen et al., 2002b; Frank-Kamenetsky et al., 2002).</p><p>Our observations that extraciliary Smo activity can regulate Gli proteolytic processing and activation suggest that a direct engagement of Smo with a signaling complex found within the primary cilium is not a pre-requisite for these biochemical events. Our findings however cannot rule out the possibility that pathway components presumed to function within the primary cilium may also act elsewhere in the cell to transduce SAG- or mutation-induced Smo activity (Briscoe and Therond, 2013; Nozawa et al., 2013). The Smo target protein Gpr161 when genetically ablated results in Gli activation irrespective of whether or not Smo is present (Mukhopadhyay et al., 2013). Thus, the ciliary depletion of Gpr161 in cells treated with SAG and IHR-1 likely contributes to Gli activity observed under these settings. Conceivably, IHR-1 and IHR-NAc could induce different Smo conformations that influence Gpr161 behavior that is not reflected by Smo ciliary accumulation and Gli activity read-outs alone. This hypothesis could be tested in the future with a greater understanding of how Smo communicates with Gpr161 and biochemical tools for monitoring their signaling activities. At the same time, we note that the number of subcellular compartments capable of supporting GPCR activity outside of the plasma membrane continue to expand as technologies to monitor their activity improve (Vilardaga et al., 2014). Future studies relying on faithful biochemical reporters of endogenous Smo activity when they become available could be leveraged to visualize the subcellular compartments of Smo activity outside of the primary cilium plasma membrane.</p><p>Our ability to bypass Smo ciliary localization for Gli regulation using a Smo agonist or activating Smo mutations raises the question of why Smo accumulation in the primary cilium is important for Hh-dependent signaling. Given the resemblance of the Hh receptor Ptch to small molecule transporters (Taipale et al., 2002) and Smo sensitivity to a variety of small molecules including those akin to cell produced lipids (Nachtergaele et al., 2012), one possibility is that Smo ciliary accumulation gates its interaction with an endogenous ligand (see Figure 6). Indeed, several studies provide evidence for differences in the abundance of certain bioactive molecules found in the primary cilium and the plasma membrane (Bielas et al., 2009; Ha et al., 2014; Jacoby et al., 2009; Janich and Corbeil, 2007; Jin et al., 2014; Tyler et al., 2009). We hope the findings presented here will galvanize efforts to identify an endogenous Smo ligand and contribute to a framework for investigating candidate molecules that emerge from such efforts.</p><!><p>The GliBS Hh pathway reporter (provided by P. A. Beachy), 8xCBP Notch pathway reporter (provided by R. Kopan), and STF Wnt/β-catenin pathway reporter (provided by R. Moon) were previously described. Smo-myc was constructed in the pcDNA3 backbone using PCR-based cloning. Smo-M2-myc and SmoL412F-myc were generated from the Smo-myc backbone using PCR-based mutagenesis. The Frizzled4 (Fzd4) expression construct was purchased from Open Biosystems. BD-cyclopamine was kindly provided by J. K. Chen. Other Smo modulators used in this study include Cyclopamine (Logan Natural Products), SANT1 (Sigma), and SAG (Alexis Biochemicals). Vismodegib was synthesized by C. Chen. ShhN conditioned medium was prepared as previously described (Chen et al., 2002b). 3T3-ShhFL and L-Wnt-STF cell lines were previously described (Chen et al., 2009; Jacob et al., 2011). Caco-2, NIH-3T3, Shh LightII (referred to as LightII cells), C3H10T1/2, Cos-7, and HEK293 cells were purchased from ATCC. Cell lines provided by other labs are listed as following: FLAG-Gli2 and SMO−/− cells (J.K. Chen), A1::Smo::GFP (referred to as Smo-GFP cells; A.P. McMahon), and PTCH1−/− (M.P. Scott). For establishing the SmoM2-myc-NIH-3T3 cell line, Smo-M2-myc cDNA was transfected into NIH-3T3 cells and clones were selected in the presence of 400ug/ml Geneticin. Subclones were then isolated from positive clones identified by Western blot analysis for SmoM2-myc expression. SmoL412F-myc cell line was established in C3H10T1/2 cells as described above.</p><!><p>For the primary screen and dose-response test, 7,000 3T3-ShhFL cells in DMEM/3% calf serum (CS) were seeded into 384-well plates and incubated at 37°C for 2hrs. 200K compounds from UT Southwestern chemical library were added into each well by a Biomek FX liquid handler (Beckman Coulter). Luciferase activities were measured by Dual-Luciferase reporter assay kit (Promega) 72hrs after drug treatment. To test the effects of compounds of interest in Wnt signaling, 5,000 L-Wnt-STF cells were seeded into 384-well plates containing diluted compounds. Luciferase activities were measured 48hrs after drug treatment. Similarly, compounds were tested in the Notch pathway using the 8xCBP reporter in cells transfected with Notch intracellular domain (NICD). For the exogenous Hh test, 12,000 LightII cells were seeded into 96-well plates. After 24hrs, culture medium was replaced with ShhN conditioned medium/3%CS. Luciferase activities were measured 48hrs after drug treatment. For testing the activity of IHR compounds against SmoM2 and SmoL412F-induced Hh pathway activity, SmoM2 DNA, SmoL412F DNA, the GliBS reporter, and a constitutive Renilla luciferase reporter were transfected into NIH-3T3 cells using Effectene (QIAGEN). For measuring the Hh pathway IC50 of IHR-1 in PTCH1−/− cells, GliBS and Renilla luciferase reporter were transfected into PTCH1−/− MEFs using Effectene. To measure IC50s of IHR compounds against SAG-induced Hh pathway activation, LightII cells were seeded into 96-well plates. For all the reporter assays described above, culture media were replaced with DMEM/3%CS that were pre-mixed with compounds after cells reached 100% confluency. Luciferase activities were measured 48hrs after drug treatment. For alkaline phosphatase assays in C3H10T1/2, cells treated for 5 days in DMEM/3%FBS in the presence of IHR compounds were lysed and alkaline phosphatase activity measured using the SensoLyte pNPP Alkaline Phosphatase Assay Kit according to manufacturer's instructions.</p><!><p>For analyzing Gli2 and Gli3 processing, FLAG-Gli2-expressing cells or NIH-3T3 cells were grown to confluence in 6-well plates. Culture medium was switched to ShhN conditioned medium or 100nM SAG in the presence or absence of Smo antagonists in DMEM/3%CS. After 48hrs of treatment with chemicals, cells were lysed in RIPA buffer. For testing Sufu and Gli3 interaction, the same protocol was followed with the exception of cell lysis using PBS with 1%NP40 after 24 hrs of chemical treatment. For Sufu immunoprecipitation, Sufu antibody was cross-linked to protein A agarose beads with dimethyl pimelimidate crosslinker (Thermo Scientific Pierce). For all the assays described above, whole cell lysate was resuspended in SDS sample loading buffer and heated to 95°C for 1min prior to SDS-PAGE. Antibodies used for analyzing the blots were: Gli3 (R&D System, AF3690), Flag epitope-tag (Sigma, F1804), PI3K-C2α (BD Transduction Laboratories, 611046), Sufu, α-tubulin and GAPDH (Cell Signaling Technology, 2522S, 2125S and 5174S, respectively).</p><!><p>For Gpr161, Smo or Gli2 cilia localization assays, IMCD3, NIH-3T3, Smo-GFP, Smo-M2-myc, or SmoL412F-myc cells were grown to confluence on 12mm poly-L-Lysine pre-coated glass coverslips (BD Biosciences). The indicated compounds were dissolved in DMEM/3%CS and then applied to cells for 48hrs or 72hrs. Cells were either fixed in 100% methanol for 5min at −20°C or in 3.7% formaldehyde for 15min at room temperature. The cells were further permeabilized in blocking buffer (5% normal goat serum and 0.2% Triton X-100 in PBS) for 10min. Coverslips were treated with anti-Smo (provided by P. A. Beachy), anti-Myc (Cell Signaling Technology, 2272), anti-GFP (MBL International, 598), anti-Gli2 and anti-Gpr161 (provided by S. Scales, Genentech), and anti-tubulin (Sigma, T6793) in blocking buffer each for 30min. After several PBS washes, the coverslips were further incubated with Alexa Fluor 488-conjugated goat anti-rabbit, Alexa 594-conjugated goat anti-mouse, or Hoechst 33342 (Invitrogen) for 30min. 100 primary cilia were scored for the presence of Gpr161, Smo, SmoM2, SmoL412F, and Gli2 in each experiment.</p><!><p>Fzd4 or Smo-myc DNA was transfected into Cos-7 cells (BD-cyclopamine binding assay) or HEK293 cells (IHR-Cy3 binding assay) by using Fugene6 in 6-well plates. 2 days after transfection, cells were incubated with medium contain fluorescently labeled compounds and the indicated competing compounds for 1hr at 37°C. Cell pellets were collected after trypsinization and washed with cold PBS three times. Cells resuspended in cold PBS were analyzed by flow cytometry on a FACS Calibur (BD Biosciences). 10,000 cells were sorted for each sample. A fluorescent positive gate was selected in areas where minimum fluorescent positive cells were detected in Fzd4 DNA transfected cells.</p><!><p>Unless otherwise noted, error bars represent standard error of the mean (SEM).</p><!><p>Synthesis of IHR-1, IHR-Cy3, and IHR-NAc is described in Supplemental Experimental Procedures.</p><!><p>Caco-2 cells were grown to confluence in 12-well transwell plates (Corning, 3460). Cell confluency was measured by Millicell-ERS volt-ohm meter (Millipore). Caco-2 cell monolayers with transepithelial electric resistance (TEER) values greater than 950 ohmxcm2 were used for experiment. Culture medium was replaced with 10μM of compounds in DMEM/3%CS and incubated for 6hrs. Media from the bottom chamber were collected and drug concentrations were determined by mass spectrometric analysis as described below.</p><!><p>Studies were executed by Cyprotex (MA). Compounds were dissolved in PBS to a final concentration of 10μM. A volume of 300μl of the 10μM compounds was added to the donor well of pre-coated PAMPA plates (BD Biosciences, 353015), and the acceptor well was filled with 200 μl of PBS. The plate was incubated for 5 hrs at 37°C. LC/MS/MS was used to analyze the samples of the donor and acceptor wells. The effective permeability (Pe) was calculated using the following equation:
Effective permeability(Pe):logPe=log{−VDVA(VD+VA)Atln(1−[drug]A[drug]E)}
VD and VA are the volumes of the donor and acceptor compartments, A is the area of the membrane, t is the incubation time, and A and E subscripts on the concentration term refer to the acceptor and equilibrium concentrations, respectively.</p><!><p>100μl of each sample was mixed with 200μl of acetonitrile containing 300ng/ml N-Benzylbenzamide (Sigma). The samples were vortexed for 15 seconds, allowed to sit 10min at room temperature, and then centrifuged 5min at 13K rpm. The supernatant was collected and centrifuged an additional time before analysis by LC/MS/MS. Standard curves were prepared using DMEM with 3% CS spiked with known concentrations of each compound. DMEM containing 3% CS was used to establish the limit of detection (LOD) as 3 times the signal seen in these samples. In general, back-calculation of standard curve points and QC samples were within 15% of theoretical. The limit of quantification was set as the lowest point on the standard curve for which back-calculation yielded values within 15% of theoretical. Analytical methods were developed to detect IHR-1 and IHR-NAc using an Applied Biosystems/MDS Sciex 3200 QTRAP mass spectrometer coupled to a Shimadzu Prominence LC. All compounds were detected as singly charged species and one daughter ion. The following transitions were monitored: IHR-1 454.8 to 173.0 and IHR-NAc 476.1 to 173.1. N-Benzylbenzamide was used as an internal standard (transition 212.1 to 91.1). Chromatography was performed using an Agilent ZORBAX XDB-C18 column (5micron, 4.6×50mm) and the following gradient conditions: 0-1 min 5% buffer B; 1-1.5min gradient to 100% buffer B; 1.5-3min 100% buffer B; 3-3.5min gradient to 5% buffer B; 3.5-5min 5% Buffer B. For IHR-1 and IHR-NAc, Buffer A consisted of 100% dH2O+0.1% formic acid and Buffer B consisted of 100% acetonitrile+0.1% formic acid.</p><!><p>An shRNA construct with the Pik3-C2α sequence 5′-GGCAAGATATGTTAGCTTT-3′ was purchased from Thermo Scientific. Pik3-C2α shRNA lentivirus was produced in 293T cells. Filtered and concentrated virus-laden medium was incubated with NIH-3T3 cells for 48hrs. Infected NIH-3T3 cells were selected with puromycin (2mg/ml) for 7 days. Cells with no more than 3 passages following were used for experiments.</p><!><p>3T3-ShhFL cells were grown to confluence in 6-well plates. Culture medium was replaced with IHR compounds (2.5μM) in 3%CS and incubated at 37°C for 48hrs. For cDNA preparation, trizol RNA extraction and ProtoScript M-MuLV First Strand cDNA Synthesis Kit (New England BioLabs) were used. 100%, 80%, 60%, and 40% of cDNA were used in the PCR reactions. Primers sequences: GAPDH forward, ATCCTGCACCACCAACT; GAPDH reverse, TGCCTGCTTCACCACCTT; Ptch1 forward, ACTGTCCAGCTACCCCAATG; and Ptch1 reverse, CATCATGCCAAAGAGCTCAA. For PI3K-C2α studies: Ptch1 forward GGAAGGGGCAAAGCTACAGT; and Ptch1 reverse TCCACCGTAAAGGAGGCTTA.</p><!><p>Smo-myc and SmoM2-myc DMA were transfected into Cos-7 cells using Fugene 6. 24hrs after transfection, indicated Smo antagonists were added at the described concentration. Cells were lysed in PBS with 1%NP40 and protease inhibitors 24hrs later. SDS sample loading buffer was added to whole cell lysates and heated to 50°C for 10min. Enzymatic deglycosylation of protein was achieved with 1μl of Endo H (Sigma) added to 30μl of whole cell lysate in sample loading buffer and incubating at 37°C for 4hrs. For Smo cell surface biotinylation, transfected Cos-7 cells were washed with ice-cold PBS, and incubated in ice-cold PBS containing 2mM Sulfo-NHS-SS-Biotin (Pierce) for 30min. Biotinylation was quenched by washing and incubating cells with ice-cold PBS containing 100mM glycine for 10min. Cells were then lysed in PBS with 1%NP40 and protease inhibitors. Agarose beads cross-linked to streptavidin (Sigma) were used to pull down biotinylated proteins. Bound proteins were recovered in sample loading buffer and separated by SDS-PAGE.</p><!><p>pBSK or SmoM2-myc were transfected into Cos-7 cells using Fugene6 in 6-well plates. Two days after transfection, the cells were washed with PBS twice and were incubated with 1ml of 10% dialyzed FBS in L-methionine and L-cysteine free DMEM. After 1hr incubation at 37°C, the cells were pulsed with 1000μCi/ml of S35-Met/Cys protein labeling mix (NEG072014MC, Perkin Elmer) for 15min. Cells were washed with 3xPBS before replacing the medium with 2ml of 10%FBS in the presence or absence of 10μM of IHR-NAc. At different chasing time point, cells were lysed in PBS/1%NP40/protease inhibitors and Smo immunoprecipitated using an anti-Myc (Santa Cruz, SC-40) antibody.</p>