Moore, Bhaskar, Gao, et al. combine live imaging of the mouse epidermis and machine learning to study the role and regulation of calcium signaling within the stem cell layer. They find that cells in G2 are necessary for coordinated tissue-wide communication and reveal a feedback loop between cell cycle and calcium signaling. 2+ signaling. We show that basal cells display dynamic intercellular Ca2+ signaling among local neighborhoods. We find that these Ca2+ signals are coordinated across thousands of cells and that this coordination is an emergent property of the stem cell layer. We demonstrate that G2 cells are required to initiate normal levels of Ca2+ signaling, while connexin43 connects basal cells to orchestrate tissue-wide coordination of Ca2+ signaling. Lastly, we find that Ca2+ signaling drives cell cycle progression, revealing a communication feedback loop. This work provides resolution into how stem cells at different cell cycle stages coordinate tissue-wide signaling during epidermal regeneration.Skin homeostasis is maintained by stem cells, which must communicate to balance their regenerative behaviors. Yet, how adult stem cells signal across regenerative tissue remains unknown due to challenges in studying signaling dynamics in live mice. We combined live imaging in the mouse basal stem cell layer with machine learning tools to analyze patterns of Ca Each day our bodies make and lose billions of cells . This re2+ signaling —to analyze and model signaling dynamics both locally between neighboring cells and at a tissue-wide level. Using this approach together with mouse models, we show that Ca2+ signaling is coordinated at large distances across thousands of cells. We find that cells in the G2 phase of the cell cycle are necessary for normal levels and patterns of Ca2+ signaling. Further, we demonstrate that most other cells in the basal layer (G1 cells) have high junctional Cx43 levels that allow Ca2+ signals to be coordinated globally, across neighborhoods of cells. Finally, we interrogate the function of coordinated Ca2+ signaling within the stem cell layer, finding that it promotes cell cycle progression, revealing a feedback loop between cell cycle and Ca2+ signaling. Together, our results provide insight into the relationship between the cell cycle in the stem cell pool and Ca2+ signaling, and how coordinated Ca2+ signaling helps maintain tissue homeostasis.To address this, we evolved our two-photon microscopy system  to captusor mice , allowin2+ signaling, which is essential for regenerative behaviors, such as proliferation, in other epithelial contexts  and combined this with a nuclear marker (K14-H2B-mCherry). Live imaging of the basal stem cell layer  layer directly above showed no signaling activity . We then quantified the number of connected nodes (cells) in each clustered signaling neighborhood and explored the temporal dynamics for each neighborhood size. Using this approach, we found that most events within the stem cell layer were either single cells that spiked in isolation from their neighboring cells  for 30 min and revisited the same region 24 h later. During a 24-h period, many cells in the basal layer progress through different cell cycle phases, and this population of cells is dynamic  was significantly lower than the signaling correlation of a region at 0 and 30 min  expresses the early differentiation marker keratin 10 .Understanding in vivo signaling dynamics of any molecular pathway at a large scale is a formidable challenge, especially in highly dynamic regenerative tissues due to both the spatiotemporal heterogeneity of signaling dynamics as well as the inherent complexity of the tissue . To analyze signaling dynamics in our system, we developed an unsupervised machine learning method called Geometric Scattering Trajectory Homology (GSTH), which captures spatial and temporal signaling patterns in a highly applicable manner and allows us to compare the Cae scales Preprint2+ signaling using GSTH time embeddings, and secondly, to understand cell-specific heterogeneity in Ca2+ signaling patterns. We elaborate on the intuitiveness and interpretability of the PHATE plots for both use cases below.GSTH produces 3D plots, which show a low-dimensional representation of the scattering transform, i.e., the concatenated zeroth-, first-, and second-order scattering coefficients, computed using the raw signaling data . This reTo quantify temporal dynamics, we compute scattering coefficients for each timepoint by aggregating over cells in the scattering transform , step 2aH0) measures the connectivity between the points, whereas dimension 1 homology (denoted H1) tracks the presence of loops in the trajectory. H0 distinguishes between “continuous” and “discontinuous” (large gaps between successive timepoints) trajectories. Discontinuous trajectories arise in datasets with spontaneous, “bursty,” or uncorrelated signaling activity. A persistent topological loop (H1) is created when a section of the trajectory takes the shape of a closed curve, with two nonconsecutive timepoints close to each other in the PHATE plot. These loops indicate the presence of quasi-periodic activity in the tissue. Conversely, persistent loops are absent from trajectories that are not as spatiotemporally organized, while many non-persistent loops are observed, indicating a scattered point cloud devoid of any trajectory-like structure.Next, we use persistent homology to quantify the shape of the trajectory in the PHATE plots. Dimension 0 homology  showed values close to 0 or 1 indicating distinct signaling dynamics in K10-reporter-positive compared to -negative cells  cells displayed different spatiotemporal signaling characteristics than their K10-reporter-negative stem cell neighbors, we used our GSTH-based Cell Embeddings method , bottom.ve cells . The aveulations . Therefo2+ signaling we observed here during homeostasis occur at random or are coordinated with one another is unclear. We applied GSTH  features . Our analysis with GSTH revealed markedly discontinuous, scattered time trajectories (H1 (loop) features. In the neuronal trajectory, many features appeared and disappeared at all scales, revealing a complex data geometry  and tracked mitotic events as they occurred, while also recording Ca2+ signaling. Surprisingly, while extensive literature links transient elevations in Ca2+ with important steps of mitosis  in epidermal basal cells, and combined this with a constitutive Ca2+-sensor . Through timelapse recordings, we observed a marked decrease in the total amount of Ca2+ signaling in G2-depleted Cdkn1b mice compared with littermate controls  were formed and closed at later thresholds, reflecting the smoothness of the time trajectory and spatiotemporal coordination of the signaling, like the controls. We next quantified Wasserstein distances (a way to measure differences in topology between trajectories) between the MMC, Cdkn1b, and control trajectories . Unlike mice enriched for G1 or mitotic cells, we observed normal spatiotemporal patterns of Cacontrols . GSTH alcontrols . To quancontrols . The G2-ectories . The Was2+ signaling in this way. As a starting point, we analyzed single-cell RNA sequencing data  and found that stem cells display a gradient of junctional Cx43 expression as they pass through different cell cycle phases, with maximal junctional localization in the G1 stage  with a germline recombined Ca2+-sensor line  and performed live timelapse imaging at 1, 5, and 7 d after induction. We first confirmed loss of Cx43 protein expression within 5 d of recombination , with topical thapsigargin treatment. Depletion of ER Ca2+ stores leads to activation of store-operated calcium entry (SOCE) as the stromal interaction molecule (STIM) senses depletion and opens the Orai Ca2+ channel  with thapsigargin. We quantified the number of green cycling S, G2, and M cells across large regions, finding that thapsigargin treatment increased the number of cycling cells just 8 h after treatment  mice  with a blue LED light or the two-photon microscope’s 920 nm Vision laser for 1 h , or in vitro models to study Caell pool . In thisuse skin  in combi2+ signaling neighborhoods across the whole-stem cell compartment. Finally, we uncovered a functional role for coordinated Ca2+ signaling, revealing that it acts as a positive feedback loop for cell cycle progression within the stem cell compartment.We identified two levels of regulation that coordinate local signaling patterns within the stem cell compartment and enable directed signal flow at long distances. First, we observed that G2 cells are necessary for coordinated signaling activity. Second, we found that neighboring G1 cells use Cx43 gap junctions to connect Ca2+ signaling within the basal stem cell compartment reinforced the existence of Ca2+ signaling activity within this pool of cells during homeostasis without perturbation  in epithelial sheets that are often a focus of attention in other systems.Our comprehensive quantification of local patterns of Caurbation . These f2+ signaling data from the basal stem cell pool, uncovering an emergent property of long-range spatiotemporally coordinated information flow. Our applications of GSTH to explore and understand dynamic cellular signaling open a new realm of possibilities for understanding not only the molecular signaling pathways that underpin regenerative processes in homeostatic and perturbed tissue environments but also any cellular signaling system beyond tissue regeneration.Further, we were able to investigate the spread of information across multiple scales by developing GSTH. Importantly, GSTH, a data-driven, unsupervised approach, is highly versatile and can be applied in the future to imaging data with any molecular sensor and across tissues with widely different geometries. GSTH revealed smooth trajectories over time for Ca2+ signaling, although they display subtly different spatiotemporal dynamics. These results demonstrate that the basal layer, as the stem cell compartment, is cohesive in its signaling dynamics and long-range communication.Our study characterizes the constituent states of the cells within the stem cell layer, finding that stem cells communicate cohesively with their differentiation-committed neighbors, as both populations remain competent to proliferate and respond to tissue demands. Using a new genetic reporter for an early differentiation marker (K10), we show that K10-reporter-positive differentiation-committed cells and K10-reporter-negative stem cells participate equally in Ca2+ signaling has been linked to cell proliferation in developmental and regenerative contexts -tdTomato-WPRE  and given doxycycline (1 mg ml−1) in potable water with 1% sucrose between P45 and P60. Doxycycline treatment was sustained until imaging was performed 3 d later. Siblings without the tetO-Cdkn1b allele  were used as controls. To visualize Ca2+ dynamics in K10+ differentiation-committed cells, Rosa26-CAG-GCaMP6s; K10-rtTA; Rosa26-Col1a1-tetO-H2B-mCherry mice were given doxycycline (1 mg ml−1) in potable water with 1% sucrose between P45 and P60. Doxycycline treatment was sustained until imaging was performed 4 d later.Germline recombined Mice from experimental and control groups were randomly selected from either sex for live imaging experiments. No blinding was done. All procedures involving animal subjects were performed under the approval of the Institutional Animal Care and Use Committee (IACUC) of the Yale School of Medicine.2, respectively, at 800 Hz or through a 25× water-immersion lens  and scanned with a field of view of 486 µm2 at 800 Hz. Z-stacks were acquired in 0.5–3 µm steps to image a total depth of up to 100 µm of tissue. Imaging data were acquired with four Hamamatsu non-descanned detectors, two GaAsP high sensitivity photomultiplier modules (Hamamatsu H7422A-40 and Hamamatsu H7422-40-LV) for green and far-red emission, and photomultiplier modules (Hamamatsu H11901-20 and Hamamatsu H6780-01) for orange and blue emission. Emission colors were separated through LaVision’s spectral detector utilizing a variety of dichroic  and bandpass filters . To visualize large areas, 2–64 tiles of optical fields were imaged using a motorized stage to automatically acquire sequential fields of view. Visualization of collagen was achieved via the second harmonic signal at 940 nm. Various fluorochromes were utilized for each experiment, including AlexaFluor 488, AlexaFluor 568, AlexaFluor 647, EGFP, mCherry, tdTomato, and mVenus. For all timelapse movies, the live mouse remained anesthetized for the length of the experiment, and serial optical sections were captured at intervals of 2 s. For revisits, the same region of live mouse skin was imaged across intervals of multiple days. Anatomical features and patterns of hair follicles and collagen were used as landmarks for finding the same skin location  for analysis. Individual optical planes, summed, or max Z stacks of sequential optical sections were used to assemble figures. To prepare movies where the nuclear signal bleached over the course of the timelapse, we used the Fiji Bleach Correction plugin , specifySegmentation of actively signaling cells was performed using the CaImAn MATLAB package . In ordeR26p-Fucci2 mice, epidermal basal cells were hand-segmented using the freehand line function in FIJI with a line width of 10 pixels to account for only the punctate Cx43 signal around each cell. Cx43 fluorescence intensity was measured as the mean fluorescence intensity in each defined circle.To analyze Cx43 immunofluorescence staining in whole-mount tissue from r = 1.0. Negative correlation coefficients mean that the signal in one image correlates with the absence of the signal in the second. Zero correlation is no correlation . A Python script module, run through FIJI, was used to calculate the 2D correlation coefficient between two images.Image correlation coefficients were calculated between two images to compare the similarity between the images . Positiv−1 Dispase II solution  at 37°C for 15 min and the epidermis was separated from dermis using forceps. The epidermis was fixed in 4% paraformaldehyde in PBS for 15 min at room temperature, washed, and blocked with 0.2% Triton X-100, 5% normal donkey serum, and 1% BSA in PBS. The samples were then incubated with primary antibodies for 12 h at 4°C and with secondary antibodies for ∼2 h at room temperature. Primary antibodies used were as follows: purified mouse anti-Connexin 43, C-terminal, clone P4G9 , rabbit anti-Connexin 30.3 polyclonal antibody , rabbit anti-Connexin 26 polyclonal antibody , rabbit anti-Connexin 31 polyclonal antibody , guinea pig anti-K10 , and rabbit anti-pH3 . All secondary antibodies used were raised in a goat host and were conjugated to AlexaFluor 488, 568, or 647 (Thermo Fisher Scientific). In some cases, tissue was then incubated with Hoechst 33342  for 15 min and then washed with blocking solution. Finally, the tissue was mounted with Vectashield Anti-fade mounting medium (Vector Laboratories) or SlowFade Diamond Antifade Mountant (Thermo Fisher Scientific) and a #1.5 coverslip and imaged on a LaVision TriM Scope II as described in In vivo imaging.Ear tissue was incubated epidermis side up in 5 mg mlK14-CreER; Cx43fl/fl; mTmG mice or K14-CreER; Cx43fl/fl mice were given three doses of tamoxifen (2 mg in corn oil) 3, 4, and 5 d before imaging or tissue collection by intraperitoneal injection. To observe phenotypes of the total loss of Cx43 just 1 d after recombination, we also topically applied 0.01 mg (Z)-4-hydroxytamoxifen (4-OHT) in an ethanol-vaseline slurry to the ear of Rosa26-CAG-GCaMP6s; K14-CreER; Cx43fl/fl or Rosa26-CAG-GCaMP6s; K14-CreER; Cx43+/+ mice 1 d before the start of imaging.To induce the expression of membrane-GFP and/or loss of Cx43 expression, −1 stock solution in dimethyl sulfoxide (DMSO) and then diluted 100 times in 100% petroleum jelly . 100 μg of the mixture of the MMC and the petroleum jelly was spread evenly on the ear 1 and 2 d before imaging. A mixture of 100% DMSO in petroleum jelly (1:100) was used as vehicle control. Demecolcine was used to block microtubule polymerization . 100 μg of the mixture of thapsigargin and petroleum jelly was spread evenly on the ear of the anesthetized mouse 30 min (Ca2+ imaging experiments) or 8 h (cell cycle experiments) before imaging. A mixture of 100% DMSO in petroleum jelly (1:100) was used as vehicle control.To block SERCA activity and activate SOCE, mice were treated with thapsigargin or DMSO vehicle control. Thapsigargin was dissolved in a 25 mg mlK14-CreER; Cx43fl/fl mice 5 d after tamoxifen induction and matching controls were fixed in a buffer. Samples were postfixed with 1% osmium tetroxide, dehydrated through a graded series of ethanol, and embedded in Epon (Electron Microscopy Sciences). Thin sections (70 nm) were cut on a Leica EM UC7 and collected on formvar-coated slot grids (Electron Microscopy Sciences). Sections were stained with 5% Uranyl acetate in 50:50 methanol:water, rinsed, and then stained with lead citrate, ready to use (Electron Microscopy Sciences). They were imaged with a Hitachi H-7650 transmission EM at 80 kV.Ear skin tissue from 2+ signaling pathway genes between different cell cycle stages, we used a previously published integrated single-cell RNA-sequencing dataset consisting of interfollicular epidermal cells from mouse dorsal skin  annotation database’s Ca2+ signaling pathway genes, plus some select genes from the literature, to create our Ca2+ signaling pathway gene list.To compare gene expression of Casal skin . In briesal skin . For subsal skin , log-norK14-CreER; Rosa26-CAG-LSL-ChR2; n = 4) mice for the cell cycle experiment, we anesthetized them along with littermate controls  with 100 μl per 12 g of body weight of rodent cocktail  10 d after receiving three intraperitoneal injections of tamoxifen (2 mg in corn oil per day for 3 consecutive days). We then placed them in a cage with a strip of blue LED lights 1 cm from their ears for 60 min. To activate ChR2  mice for the Ca2+ imaging experiment, we injected them along with littermate controls  with three intraperitoneal injections of tamoxifen (2 mg in corn oil per day) for 3 consecutive days. A week later, we simultaneously imaged and activated these mice with the two-photon microscope’s 920 nm Vision laser.To activate Channelrhodopsin-2 (ChR2)  mice and littermate controls  24 h after exposure to blue light for flow cytometry with a protocol adapted from previously described methods  at a final concentration of 2.5 µM in HBSS. The cells were stained for 30 min protected from light at 37°C and then immediately run on a Becton Dickinson LSRII outfitted with Diva software v8.0.1, and the data were analyzed using Flowjo v10.6.2.b.Epidermal single-cell suspensions were prepared from ears of ChR2  with multiple comparison’s test. Differences between the groups were considered significant at P < 0.05 and the data are presented as means ± SD, except for the violin plots, which show the median and quartiles as dashed lines. Data distribution was assumed to be normal, but this was not formally tested.Statistical analyses were performed using GraphPad Prism (version 9.2) software . Statistical parameters are reported in figure legends. Statistical comparisons were made using an unpaired two-tailed Student’s Preprint), based on the graph wavelet transform , which describes the periodicity rather than the time- or space-specificity of signals. With the prevalence of graph-structured data, there is an emerging field of graph signal processing , in whicransform , to captransform , top, wh2+ is regarded as a static node signal for each timepoint. For the epithelial cells, we defined a nearest-neighbors graph G = {V,E} with each vertex vi ϵ V being a cell, and an edge  ϵ E if the cells vi and vj are spatially adjacent. The connectivity of this cell graph G can be described by its adjacency matrix A, where Aij = 1 if vi and vj are connected  ϵ E) and 0 otherwise  representing the level of Ca2+ signaling at time t. From here on, this graph is referred to as the cellular graph.The first step of GSTH consists of defining a graph on which the Catherwise , step 1.A and D are the graph adjacency matrix and degree matrix, respectively, from step 1 and I is the identity matrix. This diffusion operator computes the probability of a lazy random walk, starting from any vertex and ending in another vertex in t steps, where each step (from one vertex to another) has a probability proportional to its adjacency and inversely to the degree. Hence, the t-th power of R, Rt, represents the probability distribution after t steps. Based on this operator R, we can then define a graph wavelet of different scales as follows  at different orders, which yields a collection of scattering coefficients S(x(t)) as overall cellular signaling pattern embeddings for timepoint t. Specifically, the zeroth-order scattering transform calculates the local averaging of raw signals x(ti) at vertex/cell vl and is obtained by:By using multiple wavelet scales Ψti are then concatenated to form the zeroth-order scattering coefficients ti. While the diffusion operator R provides local averaging of neighboring cell patterns, it also suppresses high-frequency information. We can further calculate the first-order scattering coefficients to retrieve fine-grained descriptions of high-frequency information. This is achieved by applying graph wavelets described above:The resulting scattering coefficients Similarly, the zeroth-order and first-order scattering coefficients can be further complemented by the second-order scattering coefficients:ti.Finally, we concatenate the zeroth-order, first-order, and second-order scattering coefficients to form the timepoint embedding S(X(t)) from step 2 into a low-dimensional trajectory E, where ei corresponding to a low-dimensional PHATE embedding of timepoint ti. This is achieved by applying PHATE, a dimensionality reduction method that captures both local and global nonlinear structures by constructing a diffusion geometry (S(X(t)) for each timepoint into the 3D PHATE embedding coordinates In the third step, we reduce the descriptors of signaling patterns of each timepoint geometry . The advignaling , step 3aE obtained in step 3 , we quantify their shape using persistent homology, a topological data analysis method  for each connected component (for instance) that is created at threshold ϵi and destroyed  at threshold ϵj. Persistent homology is still under-explored in data science but offers a way of quantifying general shape features of datasets. For instance, we can differentiate between smoother trajectories  versus rougher trajectories . Moreover, dynamic trajectories from different mice can be readily compared using well-defined distance metrics between the corresponding persistence diagrams. Here, we use the 2-Wasserstein distance  for the persistence diagram in dimension q. More precisely, the Betti curve of dimension q of a diagram Q refers to the sequence of Betti numbers, i.e., active topological features, of dimension q in Q, evaluated for each threshold ϵ. It is a useful descriptor for numerous machine-learning tasks  and, by extension, of the Ca2+ signaling data.A second way to visualize the results is through the so-called Betti curves that are associated with a persistence diagram ng tasks . Intuiti2+ signaling patterns of different individual cells within the same experiment , based on all points in time, to compare the Caperiment , bottom.vi across all timepoints as features  and revisits (hours–days). 2+ signaling patterns in the basal stem cell layer reveals coordination and directed spread of signals across time. 2+ signaling. 2+ signaling. 2+ signaling. 2+ signaling across the basal stem cell layer. 2+ signaling. 2+ signaling. 2+ signaling. 2+ signaling. 2+ signaling at large scales, but not across local neighborhoods, in the stem cell pool. Table S1lists all the genes analyzed in the scRNAseq dataset.Click here for additional data file.


UA is used in animal husbandry as an anti-inflammatory and antiviral agent, as well as for enhancing the integrity of the intestinal barrier. Although UA has been shown to have significant in vitro bacteriostatic effects, it is rarely used in animal nutrition. The use of UA as a substitute for oral antibiotics or as a novel feed additive in animal husbandry should be considered. This review summarizes the available data on the biological functions of UA and its applications in animal husbandry.Ursolic acid (UA) is a plant-derived pentacyclic triterpenoid with 30 carbon atoms. UA has anti-inflammatory, antioxidative, antimicrobial, hepato-protective, anticancer, and other biological activities. Most studies on the biological functions of UA have been performed in mammalian cell ( UA contnosa L.) . DetailsIn terms of biopharmaceutical classification, UA is a Class IV compound (“low” solubility and “low” permeability) . The cliUA has been proposed as a candidate drug for treating various cancers , inflamm2.2.1.in vivo and in vitro studies have shown that UA acts against both endogenous and exogenous inflammatory stimuli, with favorable anti-inflammatory effects. UA  can prevent the degeneration of dopaminergic neurons in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinsonian mice (in vivo as well as protect against lipopolysaccharide (LPS)-induced acute kidney injury by blocking the Toll-like receptor/myeloid differentiation primary response 88 pathway in vitro -treated A549 cell model, resolved both cell injury and the inflammatory response . Zhou an2.2.in vitro antioxidant activity of UA was evaluated by inhibiting 2,2-diphenyl-1-picryl-hydroxyl at an inhibitory concentration (IC50) of 59.7 ± 1.0 μg/mL -NF-κB pathway (4)-induced liver fibrosis (Oxidative stress results from excessive production of reactive oxygen species (ROS) in cells and tissues, which further leads to various inflammation-associated diseases . The in .0 μg/mL . Adminis pathway . In huma pathway . Further pathway , includi pathway , obesity pathway , liver dfibrosis , and ostfibrosis .NFE2L2, is a regulatory factor of phase II antioxidant enzymes, protecting the body from oxidative stress and inflammation. Nrf2(−/−) mice showed obvious symptoms of neurodegeneration and oxidative stress, which were significantly reduced by intraperitoneal injection of UA (100 mg/kg), demonstrating the neuroprotective effects of UA (4-induced hepatic oxidative damage and inflammation by increasing the expression levels of NAD(P)H:quinone-oxidoreductase-1, glutathione S-transferases, heme oxygenase-1, B-cell lymphoma-2 (Bcl-2), and nuclear Nrf2  showed antioxidant activity by upregulating the expression of peroxiredoxin-2 and skn-1 transcription factor (which corresponds to human Nrf2); this also led to an anti-depressant effect  pathway . Nrf2, ets of UA . UA  values of 12.8 μM, 7.7 μM, 16.8 μM, and 19 μM, respectively (In vitro experiments confirmed that 48 h of UA treatment (16 μM) could reduce the adhesion and infiltration of the human breast cancer adriamycin-resistant cell line MCF-7/ADR to human umbilical vein endothelial cells (HUVECs) and could also reduce migration in MCF-7/ADR cells  signaling pathway, respectively, and downregulates the expression of the apoptosis regulator Bcl-2 . In a prectively . Furtherectively . Furtherectively . These rectively . In addiectively . UA alsoDR cells . UA induDR cells .2.4.in vitro experiments also confirmed that UA (20 μM) significantly reduced TC (37.2%) and TG (50.4%) contents in HepG2 cells and upregulated P-AMPK protein expression  and triglyceride (TG) levels in the liver and plasma, effectively relieve liver steatosis and reduce the number of epididymal fat cells. Meanwhile, pression . Therefossion /ROS signaling pathway in hepatic stellate cells , Enterobacter cloacae ATCC-BAA 2468, and Pseudomonas aeruginosa ATCC 25000 , increasing the sensitivity of ibiotics . UA , and inhibited biofilm formation of carbapenem-resistant Klebsiella pneumoniae (E. coli (ATCC 25922), K. pneumoniae (ATCC10031), and Shigella flexella (ATCC12022) was 64 μg/mL. However, the MIC of UA against these bacteria was significantly reduced by modification of C-3 (hydroxyl) in UA to form UA ester analogs. Hence, the modified UA derivatives showed markedly increased antibacterial effects , Pseudomonas aeruginosa, and Vibrio havii, respectively, after culture for 24 h. Furthermore, 10 and 30 mg/L of UA induced the expression of genes related to chemotaxis . Overexpression of the motAB genes has been shown to cause increased bacterial activity, decreasing their stability in the biofilm environment and resulting in reduced biofilm formation  also exerts the cytotoxicities against A549 (lung cancer cell line) and NTUB1 cells (human bladder cancer cell line)  could inhibit the spermatogenesis in Wistar strain male albino rats (3 months old) . 50 μM Ull line) . In the 26 g/ kg .4.At present, the application of UA in animal husbandry is limited. UA is mainly used to improve antioxidant capacity in broilers  and as aRosmarinus officinalis L., Aurantii fructus immaturus, and Eucommla ulmoides leaves) for 42 days resulted in a significant reduction in elimination and mortality rates of the sperm, while the malondialdehyde content was also decreased due to an increase in the activity of superoxide dismutase   .in vitro  in rainbow trout (Oncorhynchus mykiss). When UA was injected intraperitoneally, a reduction in the expression levels of IHNV glycoprotein mRNA (in the spleen) was observed on day 1 after viral infection   replication (IC50 = 5.55 μM) in vitro and increase the mortality rate by 12.5% in MSRV-infected largemouth bass  enzymes produced by E. coli  also exhibited a strong anti-H5N1 activity . Furtherin vitro . UA is onfection . In addi aurata) . UA couluth bass . This inactivity , and the E. coli . Based o5.Lavandula stoechas, apple peel, rosemary, and ligustrine. Numerous investigations have demonstrated that UA has a variety of biological activities, including anti-inflammatory, antioxidant, anti-cancer, and hepatoprotective effects, using both in vitro and in vivo animal model experiments  of UA require further investigation.GL, PQ, and XC wrote sections of the manuscript. LW and RW made the figures. WG provided the topic of this article. All authors contributed to the article and approved the submitted version.


G-protein-coupled receptors (GPCRs) are ubiquitous sensors and regulators of cellular functions. Each GPCR exists in complex aggregates with multiple resting and active conformations. Designed to detect weak stimuli, GPCRs can also activate spontaneously, resulting in basal ligand-free signaling. Agonists trigger a cascade of events leading to an activated agonist-receptor G-protein complex with high agonist affinity. However, the ensuing signaling process can further remodel the receptor complex to reduce agonist affinity, causing rapid ligand dissociation. The acutely activated ligand-free receptor can continue signaling, as proposed for rhodopsin and μ opioid receptors, resulting in robust receptor activation at low agonist occupancy with enhanced agonist potency. Continued receptor stimulation can further modify the receptor complex, regulating sustained ligand-free signaling—proposed to play a role in opioid dependence. Basal, acutely agonist-triggered, and sustained elevated ligand-free signaling could each have distinct functions, reflecting multi-state conformations of GPCRs. This review addresses basal and stimulus-activated ligand-free signaling, its regulation, genetic factors, and pharmacological implications, focusing on opioid and serotonin receptors, and the growth hormone secretagogue receptor (GHSR). The hypothesis is proposed that ligand-free signaling of 5-HT2A receptors mediate therapeutic effects of psychedelic drugs. Research avenues are suggested to close the gaps in our knowledge of ligand-free GPCR signaling. The superfamily of G-protein-coupled receptors (GPCRs) comprises ~800 gene encoding proteins with seven transmembrane domains , with ovBecause receptors are tuned to respond to minute triggers, one can expect spontaneous receptor activation to occur resulting in ligand-free signaling, common to most receptor types, including growth factor receptors that regulate cell proliferation . Many GPLigand-free signaling, also referred to as spontaneous, constitutive, or basal signaling, has been reported for diverse GPCRs ,19,20,212R) , d, d+ and  and GTP B [58]. I and GTP B.3H-etorphine sites in vivo  ). T). T66]).0 mg/kg) ,67. BothLSD is one of the most potent 5-HT2A agonists, eliciting hallucinations with an oral dose at or above 0.025 mg in humans . As discContinuing signaling of ligand-free R* after agonist stimulation could be generally applicable to many GPCRs. If ligand-free R* accounts for a main portion of the agonist’s overall effect, agonists such as etorphine and LSD can reach extreme potencies as only a small fraction of the receptor population needs to be occupied. Partial agonists such as buprenorphine would need to occupy a larger portion of available receptor sites before sufficient activated agonist-R* and ligands-free R* is generated for analgesic effects . The halO* signaling whereas neutral antagonists can bind but do not block RO* signaling , since  diverge , acting  diverge . Discrep1-blocker such as carvedilol may lead to better survival outcomes than inverse agonist [Inverse agonists or neutral antagonists can be the drug of choice at ligand-free signaling GPCRs. For example, basal activity of β-adrenoceptors has led to the development of neutral and inverse agonists with distinct pharmacological effects . For tre agonist . Modulat agonist .Antagonists can differ in their property as inverse agonist or neutral antagonist at the same GPCR as a function of the signaling pathway  or pretrR, in a ratio of 1:4  [Partial agonists are proposed to be less efficient than full agonists in generating agonist-R*, which can further convert into ligand-free R* . The parorphine) . Upon ororphine) . We had orphine) . FurtherFor the interpretation of imaging studies of GPCRs with potent labeled ligands , the rec3H-etorphine binds with high affinity to the receptor, but upon activation of signaling, appears to lose affinity at the agonist-R* state and dissociates more rapidly than shown in vitro [Antagonists appear to bind to both silent and active receptors with high affinity, thereby labeling a large pool of receptor sites. On the other hand, agonist tracers label the same receptor with at least two distinct affinities thought to reflect low affinity binding to the uncoupled receptor and high affinity binding to the active agonist-receptor G-protein complex, as discussed with serotonin receptors ,98. Yet,in vitro . Opioid in vitro . These din vitro . As a rein vitro . Similarin vitro . DistincAs ligand-free signaling of many GPCRs has physiological effects, one can assume that it is regulated by mechanisms similar to those applicable to agonist activated signaling, involving interactions with kinases, arrestins, and other regulatory factors. For example, MC4R displays basal ligand-free signaling and is involved in regulating appetite and metabolism. Its endogenous agonist α-melanocyte-stimulating hormone (α-MSH) causes anorexigenic effects, whereas its endogenous inverse agonist agouti-related peptide (AgRP) blocks MC4R ligand-free signaling with orexigenic effects , one exaO  after administration of small doses in mice and guinea pigs (unpublished data), low fractional MOR occupancy appears to be sufficient gradually to reverse the dependent state. Low-dose 6β-naltrexol also prevents neonatal withdrawal behavior in guinea pig pups born to dams exposed to methadone (IC50 ~0.025 mg/kg) [In contrast to naloxone, 6β-naltrexol does not cause withdrawal in an acute model of dependence (4 h after a single dose morphine) but blocks the effect of naloxone, indicating that endogenous opioids do not play a main role . With ch5 mg/kg) .O, as depicted in The proposed reversal of ligand-free MOR signaling had been tested previously in mice with use of afferent nociceptors that express silent MOR sites . InflammThe unique pharmacological properties of 6β-naltrexol led us to the hypothesis that 6β-naltrexol or its analogs could serve in the design of safer opioid analgesic co-formulations, weaning protocols, and prevention of neonatal abstinence syndrome ,55,110. αi1 pathway but neutral antagonists at the Gαq/11 pathway. The Gαi1 pathway is also considered the hallucinogenic effector pathway [/** states and the resting 5-HT2AO state? Critical analysis of psychedelic drugs can help address some of these questions.A key neurotransmitter, serotonin has broad physiological functions interacting with serotonin receptors encoded by 12 genes. Among these, the 5-HT2A receptor serves as a common drug target in the treatment of schizophrenia and of depression indirectly by blocking serotonin transporters (SERT and SLC6A4). Drugs targeting 5-HT2A typically interact with multiple neurotransmitter receptors of high sequence homology, confounding a clear understanding of the underlying mechanisms responsible for clinical outcomes. In addition, 5-HT2A can form heterodimers with DRD2  and the  pathway ,119. In 5-HT2A receptors are main targets for psychedelic drugs such as psilocybin, mescalin, and LSD . Acting αi1, Gq/11, or β-arrestin-1 [3H-ketanserine, EC50 76 nM in a Ca++ assay [Interactions between psychedelics with 5-HT2A receptors appear to be similar to those with opioid receptors. Structural analyses have determined the shape of ligand binding pockets and structures of the receptor with and without agonist ligands coupled to Grestin-1 ,92,119. restin-1 ,128,129.restin-1 . This hy++ assay ). TherefSingle doses of psychedelics cause hallucinations over several hours, and extended mood changes for 1–2 days ,130,131.An alternative mechanism proposed to account for the antidepressant effects of psychedelics and antidepressants involves direct binding to TRKB receptors resulting in enhanced binding of neurotrophins to TRKB  ,134, theMicro-dosing of psychedelics, at levels 5–10% of those causing hallucination, appears to suffice for attaining long-lasting effects as a potential therapy with broad applications ,137. TheGHSR mutations, the Ala204Glu GSHR variant (rs121917883) selectively impairs ligand-free signaling [GHSR, and specifically its active splice-variant GHSR1a, is the receptor for the orexigenic peptides ghrelin and acetyl-ghrelin with a broad spectrum of physiological effects, such as stimulating growth hormone release, increasing hunger, modulating energy metabolism, and regulating immune function . Expressignaling ,143. Theignaling , but in ignaling .Signaling by GHSR1a is regulated on many levels, including by endogenous ligands, the potent agonist ghrelin/acyl-ghrelin and the inverse agonist LEAP2, both affecting eating behavior in opposing directions . SimilarGHSR1a is a prolific partner, forming heterodimers with other GPCRs, including DRD1, DRD2, MC3R, and 5-HT2C, thereby allosterically modulating ligand-free signaling of its partner receptor ,37,38. LO*).Genetic variants can change ligand-free signaling of GPCRs in multiple ways. Enhanced or reduced expression will alter ligand-free signaling while enhanced receptor clustering could further promote ligand-free signaling. Changes in expression of receptor protein can result from genetic effects on transcription, RNA processing, translation, protein turnover and modifications, and cellular trafficking. Genetic variants altering protein structure and function can impact the degree of ligand-free signaling and efficacy of ligands including inverse agonists. A detailed study of genetic variants across populations has the potential to reveal insight on ligand-free signaling; yet, published studies fail to cover this spectrum of mechanisms, rather focusing on a limited set of functional assays related to basal ligand-free signaling . For example, the database lists 157, 102, and 105 missense variants for OPRM1, 5HT2A, and GHSR, respectively, including information on mutations that stabilize inactive/active states. GPCRs showing high ligand-free signaling—either natively or as a result of mutations—have been used for drug discovery [The NaVa database lists natural GPCR variants , while tiscovery  and ligaiscovery . Naturaliscovery ,132,150.iscovery . Constitiscovery , adhesioiscovery , angioteiscovery , thromboiscovery , and morNaturally occurring mutants of CXCR4 that promote enhanced signaling are linked to WHIM syndrome, a rare immunodeficiency disorder ,155. LigGHSR mutations have been shown also to result in stunted growth, GHSR is a drug target for the therapy of delayed growth in pediatric patients [Loss or gain of function of endocrine receptors with functionally important ligand-free signaling has been associated with various endocrine disorders . Inappropatients .GPCRs play a key role in cancer by regulating tumor angiogenesis, immune evasion, metastasis, and drug resistance  and can This brief synopsis highlights the importance of ligand-free signaling revealed by genetic variants.O*) observed for many GPCRs, acutely activated ligand-free signaling (R*), and sustained signaling of regulated ligand-free R**  is emphasized across several GPCRs, affording new avenues to manipulate ligand-free signaling. In particular, compounds changing lasting ligand-free signaling are potential drug candidates when given at doses below those affecting the acute pharmacological response. Such slow acting, low-dose regimen can result in novel therapies exploiting the physiological relevance of ligand-free GPCR signaling.The following granted patents pertain to 6β-naltrexol and congeners: US8,883,817; 8,748,448; 9,061,024; and 10,925,870.


Prostate cancer is the second most diagnosed form of cancer in men worldwide and accounted for roughly 1.3 million cases and 359,000 deaths globally in 2018, despite all the available treatment strategies including surgery, radiotherapy, and chemotherapy. Finding novel approaches to prevent and treat prostate and other urogenital cancers effectively is of major importance. Chemicals derived from plants, such as docetaxel and paclitaxel, have been used in cancer treatment, and in recent years, research interest has focused on finding other plant-derived chemicals that can be used in the fight against cancer. Ursolic acid, found in high concentrations in cranberries, is a pentacyclic triterpenoid compound demonstrated to have anti-inflammatory, antioxidant, and anticancer properties. In the present review, we summarize the research studies examining the effects of ursolic acid and its derivatives against prostate and other urogenital cancers. Collectively, the existing data indicate that ursolic acid inhibits human prostate, renal, bladder, and testicular cancer cell proliferation and induces apoptosis. A limited number of studies have shown significant reduction in tumor volume in animals xenografted with human prostate cancer cells and treated with ursolic acid. More animal studies and human clinical studies are required to examine the potential of ursolic acid to inhibit prostate and other urogenital cancers in vivo. Prostate cancer is the second most diagnosed form of cancer in men worldwide (second to lung cancer). It is responsible for nearly 400,000 deaths annually, approximately 4% of cancer related deaths in men . ProstatProstate cancer is classified as either androgen sensitive or androgen insensitive. Androgens such as testosterone promote growth of the prostate gland. At the cellular level, the binding of androgens to androgen receptors (AR) leads to nuclear translocation of the AR–androgen complex which acts as a transcription factor activating genes promoting cell proliferation as well as the synthesis of prostate-specific antigen (PSA) . Early sThe phosphatidylinositol 3-kinase (PI3K)/Akt pathway plays a significant role in prostate cancer. Expression/activation of Akt is often elevated ,5 and abAnother characteristic of cancer cells, including prostate cancer cells, is the suppression of the process of programmed cell death known as apoptosis. Activation of both the intrinsic and extrinsic apoptotic pathways lead to the cleavage/activation of caspases and downstream cleavage of poly (ADP-ribose) polymerase-1 (PARP-1) and induction of apoptosis ,15. The Apart from androgen deprivation therapy, other treatment strategies for prostate cancer include surgery/prostatectomy, radiation therapy, and chemotherapy. The treatment approach for each patient depends on the stage of the disease. In advanced in situ carcinoma cases, surgery or radiotherapy are the preferred treatment options. Although surgery reduces the potential for metastasis, it is unfortunately not associated with a high reduction in the mortality risk after 10 years. Radiotherapy, specifically external-beam radiotherapy and brachytherapy, are effective in treating localized tumors and are associated with higher life expectancy/lower mortality risk after 10 years ,17. All Taxus brevifolia) and the needles of the European yew tree (Taxus baccata), respectively [Many drugs used in chemotherapy are derived from plants. For example, the chemotherapy drugs paclitaxel and docetaxel, used in prostate cancer treatment, were originally isolated from the bark of the Pacific yew tree  is found in the leaves and fruits of many plant species with high concentrations detected in lavender, marigold, rosinweed, basil, rosemary, and olive tree leaves. In addition, fruits such as cranberries, black elderberries, apples, and pears  contain 30H48O3, and structurally, UA is classified as a pentacyclic triterpenoid  cancers. A search was performed in PUBMED.com for the terms “ursolic acid and prostate cancer”, “ursolic acid and renal cancer”, “ursolic acid and bladder cancer”, and “ursolic acid and urogenital cancers”. Articles which specifically examined UA and these cancers were included and presented in chronological order.The treatment of human androgen-sensitive LNCaP and hormone refractory PC-3 prostate cancer cells with UA (55 µM for 24 and 48 h) reduced viability (MTT assay) and induced apoptosis (Annexin assay) and these effects were associated with a downregulation of the anti-apoptotic protein B-cell lymphoma 2 (Bcl-2)  Table 1Table 1. Zhang et al. showed that UA dose-dependently decreased viability and induced apoptosis in both LNCaP androgen-dependent and LNCaP-A1 androgen-independent prostate cancer cells . WesternLNCaP prostate cancer cells treated with UA 40 µM) had suppressed proliferation and increased apoptosis which occurred via mediation of the ROCK1/PTEN signaling pathway. UA induced cleavage of ROCK1 and phosphorylation of PTEN leading to increased protein expression of cytochrome c and cofilin-1. Increased cytochrome c due to UA treatment lead to increased activity of caspases-3 and -9 [ µM had sThe treatment of primary malignant tumor (RC-58T/h/SA#4)-derived human prostate cancer cells with UA inhibited survival, reduced cell density, and increased apoptosis. Cell treatment with UA  caused nuclear condensation, formation of apoptotic bodies, and DNA fragmentation, all markers of apoptosis. Further investigation of the cell cycle with flow cytometry showed an increased number of cells in the subG1 phase. An increase of active caspase-3, -8, and -9 was observed with UA treatment (investigated by fluorometric assays). Western blot analysis showed upregulation of the pro-apoptotic protein Bax, downregulation of the anti-apoptotic protein Bcl-2, and induction of Bid cleavage. These data suggest that UA treatment stimulates the activation of caspase-8 leading to Bid cleavage and downstream activation of caspase-9. UA also increased the expression of mitochondrial apoptosis factor (AIF) and caused its translocation into the nucleus. Together these data suggest that treatment of RC-58T/h/SA#4 prostate cancer cells with UA induces apoptosis through both caspase-dependent and independent pathways .Exposure of PC-3 prostate cancer cells to UA resulted in reduced viability and increased apoptosis through activation of both intrinsic and extrinsic apoptotic pathways. Fluorometric assays showed increased caspase-3, -8, and -9 activity while Western blotting demonstrated increased cleavage of caspase-8 and -9. Inhibiting either caspase-8 or -9  prevented the UA-induced apoptosis. In addition, UA treatment lead to activation of JNK and subsequent Bcl-2 phosphorylation. UA reduced Akt phosphorylation and increased the levels of Fas ligand (FasL). FasL knockdown by a small interfering RNA (siRNA) approach attenuated the UA-induced caspase-8 activation and apoptosis. These data suggest FasL involvement in the UA-induced apoptosis of PC-3 prostate cancer cells. Cell invasion was also inhibited as evidenced by the downregulation of the matrix metalloproteinase-9 (MMP-9). Taken together, the data of this study support UA as a potential therapeutic agent against prostate cancer due to its ability to induce apoptosis via both the intrinsic and extrinsic pathways and to inhibit invasion and metastasis .Shin et al. demonstrated that PC-3 prostate cancer cells treated with UA had reduced viability associated with cell cycle arrest at the G1 phase . In addiMeng et al. showed that prostate cancer cells LNCaP and PC-3 treated with UA had reduced proliferation and increased apoptosis . UA causAndrogen-independent DU145 and androgen-dependent LNCaP prostate cancer cells treated with UA had decreased proliferation and increased DNA fragmentation, an indicator of apoptosis [In a study by Zhang et al., human DU145 androgen-refractory prostate cancer cells were treated with UA resulting in a concentration-dependent decrease in cell viability. In addition, UA induced apoptosis as evidenced by fluorescence microscopy showing nuclear shrinkage, condensation, and fragmentation, all morphological changes typical of apoptotic cells . In thes2 transcript levels when treated with UA (25 µM) [2 led to activation of Src and phosphorylation of p38 leading to downstream overexpression of COX-2; COX-2 overexpression caused the cells to become resistant to apoptosis [2 activation and subsequent COX-2 overexpression led to UA-induced apoptosis resistance. An important finding in this paper was the dual role of UA as a cancer treatment and its ability to both induce apoptosis and create resistance to it. Having a better understanding of the mechanisms involved in apoptosis resistance is important in designing cancer drugs [DU145 prostate cancer cells had an increase in intracellular ATP and P2Y (25 µM) . Activatpoptosis . COX-2 oer drugs .Ursolic acid-induced apoptosis of DU145 prostate cancer cells is based on the increased activity of the pro-apoptotic proteins caspase-3 and caspase-9. Protein levels of cytochrome c were increased in the cytoplasm and suppressed in the mitochondria suggesting mitochondrial apoptosis pathway activation. UA in these cells suppressed the rho-associated protein kinase/phosphatase and tensin homolog (ROCK/PTEN) signaling pathway which led to the inhibition of cofilin-1 protein expression. Together, these data suggest that, in DU145 prostate cancer cells, UA induces apoptosis through ROCK/PTEN-mediated mitochondrial translocation of cofilin-1 .Park et al. also found that treatment of PC-3, LNCaP, and DU145 prostate cancer cells with UA resulted in a dose-dependent increase in apoptosis . In addiUrsolic acid treatment sensitized tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-resistant prostate cancer cells (LNCaP and DU145) allowing TRAIL-induced apoptosis to occur . UA + TRVentral prostate tumor cells derived from mice, HMVP2, were treated with 20 µM UA and a series of other natural compounds, including curcumin (CUR) and resveratrol (RES), to determine the most effective combinations . This scShanmugam et al. showed that human prostate cancer cells  treated with UA not only had reduced viability, as measured by MTT assay, but also reduced cell migration . In addiVaccinium macrocarpon (cranberries) inhibited the growth of DU145 prostate cancer cells. UA treatment reduced the activity of matrix metalloproteinases MMP-2 and MMP-9 as examined by gelatin gel electrophoresis [UA extracted from phoresis . These dIn experiments by Wang et al., prostate cancer cells were treated with UA (20 µM) or phenethyl isothiocyanate  and analyzed using qPCR and Western blotting. UA treatment resulted in changes to the mRNA levels of Setd7, Nrf2, quinone oxidoreductase 1 (Nqo1), and glutathione S-transferase theta 2 (Gstt2). In LNCaP cells, but not PC-3, UA treatment increased the protein expression of Setd7. Setd7 and Nqo1 protein expression were increased with PEITC . Short hairpin-RNA (shRNA) was used to knockdown Setd7 leading to the inhibition of colony formation (LNCaP and PC-3) and increased ROS (LNCaP cells). Chromatin immunoprecipitation assays showed that knockdown of Setd7 decreased H3K4me1 at the Nrf2 and Gstt2 promoter region and this effect was attenuated with either UA or PEITC treatments . These dDU145 prostate cancer cells treated with either UA (30 µM) or exposed to 5 Gy of irradiation had decreased survival. However, when irradiation and UA were combined, a greater decrease in cell viability and induction of apoptosis was seen. The increased apoptosis was associated with the activation of caspase-3 .Overall, the articles presented here provide strong evidence for the effects of ursolic acid against proliferation and survival and induction of apoptosis of prostate cancer cells. In addition, the treatment of prostate cancer cells with UA resulted in inhibition of JNK, Akt, mTOR, p70 S6K, 4EBP1, and NF-κB . MoreoveOnly a few studies have utilized mouse xenograft models of prostate cancer to examine the effects of ursolic acid in vivo . HMVP2 pIn another study, immunodeficient mice transplanted with human VCaP-Luc prostate cancer cells had reduced tumor growth when administered ursolic acid. Cellular metabolites and metabolism-related signaling pathways were regulated by UA including S-adenosylmethionine (SAM), suggesting potential methylation reprogramming resulting in overall anticancer effects .w/w UA continuously for 36 weeks, which initially resulted in delayed formation of prostate intraepithelial neoplasia (PIN). During weeks 12–18 of the experiment, there was an inhibited progression of the PIN to adenocarcinoma. UA treatment reduced tumor growth and prolonged overall survival without affecting body weight. Subsequent experiments revealed that UA treatment led to downregulated activity of pro-inflammatory mediators such as NF-κB, STAT3, AKT, and IKKα/β in prostate tissues and reduced serum levels of TNF-α and IL-6. Immunohistochemical analysis of tumor tissue samples revealed reduced expression of cyclin D1 and COX-2 and increased levels of caspase-3. The serum samples had nanogram levels of detectable UA. Taken together these data support the use of UA for both prevention and treatment of prostate cancer [Shanmugam et al. fed transgenic adenocarcinoma of mouse prostate (TRAMP) mice a diet containing 1% e cancer .Nude mice were injected subcutaneously with DU145 prostate cancer cells and then fed a diet containing 200 mg/kg UA twice a week for 6 weeks [Transgenic adenocarcinoma of mouse prostate (TRAMP) mice treated with UA showed suppressed CXCR4 expression in the prostate and inhibited metastasis of prostate cancer to distal organs including the liver and lungs. CXCR4 is an important signaling molecule in cancer metastasis and its downregulation with this treatment suggests UA has potential as a treatment against prostate cancer cell metastasis .A transgenic line of mice was created in which the PTEN tumor suppressor gene was specifically knocked out in prostate tissues (PTEN KO). PTEN KO mice had an age dependent increase in the size of the prostate lobes, and this increase was attenuated in mice fed a diet containing UA. Epigenomic CpG methyl-seq analysis showed that UA was able to attenuate the differentially methylated regions induced in PTEN KO mice .Furthermore, UA abrogated the PTEN KO-induced prostate cancer-related oncogenes Has3, Cfh, and Msx1. Association analysis of these studies identified a correlation between methylation status and mRNA expression of the tumor suppressor gene BDH2 and oncogenes Ephas, Isg15, and Nos2. These data suggest that UA may regulate oncogenes and/or tumor suppressor genes through modulation of their promoter methylation at an early stage of tumorigenesis. A metabolomic study was also performed and found that UA attenuated the PTEN KO-induced cancer-associated metabolic changes. UA attenuated purine metabolism/metabolites as well as glycolysis/gluconeogenesis metabolism and pyruvate and lactate levels. These changes in metabolism suggest UA has an important role in PTEN KO-mediated metabolic and epigenetic reprogramming and therefore, UA protects against PTEN knockout-induced tumorigenesis . This isFemale athymic nude mice treated with 20–40 mg/kg of UA intraperitoneally for 3 weeks resulted in a decrease in tumor volume and weight. Inhibition of proliferation and induction of apoptosis was evident. Immunohistochemical analysis revealed a decrease in p-Akt, p-mTOR, and Ki67 .A few studies also examined the effect of UA against renal and bladder cancers . ExperimGai et al. found that treatment of T24 bladder cancer cells with UA resulted in the inhibition of proliferation and induction of apoptosis. Treatment with UA resulted in inhibition of Akt and IκBα–NF-κB signaling, Bcl-2 downregulation, and caspase-3 upregulation . These dNon-toxic concentrations of UA have also been reported to pose antiangiogenic activity. Exposure to UA has been associated with decreased VEGF and iNOS expression levels in different tumor types . UA alsoUrsolic acid derivatives have been suggested to pose antitumor activity against renal and bladder cancers . A studyChadalapaka et al. reported that UA-derived analogs (2-position in the A-ring substitution) exhibited growth inhibition effects on KU7 and 253JB-V human bladder cancer cells  Table 4Table 4. Exposure of T24 human bladder cancer cells to UA resulted in a dose-dependent inhibition of growth and induction of apoptosis . InteresIn another study by the same group, exposure of T24 cells to UA resulted in inhibition of clonogenic survival and induction of apoptosis that was associated with a significant increase in endoplasmic reticulum (ER) stress as seen by the increased phosphorylation of the ER membrane receptor double-stranded RNA-activated protein kinase (PKR)-like ER kinase (PERK) and increased levels of CHOP . TreatmeLin et al. also reported that the antiproliferative and pro-apoptotic effects of a UA derivative (UA17) in NTUB1 human bladder carcinoma cells were associated with increased ROS and p53 levels, increased activation of p38 MAPK, and downregulation of Bcl-2 protein . The comHuang et al. reported that UA enhanced gemcitabine-induced cytotoxicity in T24 and 5637 bladder cancer cells in vitro. Increased activation of JNK signaling pathway and inactivation of the PI3K/AKT signaling pathway was observed . These dStudies examining the ability of UA and its derivatives to sensitize tumor cells to chemotherapeutic agents as well as to ionizing radiation have been summarized in a review by Prasad et al. .Corni Fructus in PC-12 cells in vitro [Jung et al. examined the anticancer effects of UA in preclinical models of pheochromocytoma . The invin vitro . They rein vitro . On the in vitro .Although direct evidence from preclinical studies on the effect of UA and its derivatives on human testicular cancer models is lacking, indirect evidence suggests that it might provide some benefit as a complementary treatment. Both seminomatous and nonseminomatous testicular tumors are extremely chemosensitive as well as radiosensitive . As prevIn the Biopharmaceutics Classification System (BCS), UA is classified as a type IV compound, being almost insoluble in water and exhibiting poor oral bioavailability and intestinal permeability . UA is aThe pharmacokinetics of UA have been reported both in rodents and humans. In particular, plasma concentrations and tissue distributions of UA were measured by Chen et al. using liquid chromatography-mass spectrometry (LC-MS) in Sprague-Dawley rats. An oral dose of UA (10 mg/kg) was administrated, and plasma levels peaked at 1.1 µg/mL (2.4 µM) approximately 30 min after exposure while the highest concentration of UA was found in the lung . Rats feIn humans, Hirsch et al. reported that a single oral dose of UA  had very low bioavailability in healthy adult volunteers (14 subjects) . Since t2) and the maximum plasma concentration observed was 3404.6 ± 748.8 ng/mL [2 doses, while eight patients were to a multiple-dose study and received 74 mg/m2 doses for 14 consecutive days. The results showed that the maximum plasma concentration increased linearly with dose escalation  while no drug accumulation was observed after repeated administration [A number of pre-clinical or clinical studies has been conducted the last ten years to investigate the bioavailability and pharmacokinetics of UA-related formulations which indicated that liposomes are more effective and safer compared to nanospheres and polymeric micelles . For exa.8 ng/mL . Twenty-stration .2 [2 to 98 mg/m2, indicating that they are a more effective means for delivering UA.Wang et al. enrolled 63 subjects for a single-dose study of UA liposomes, which presented linear values of pharmacokinetic parameters and showed that the maximum tolerated dose of UA liposomes was 98 mg/m2 . The sam2 . Overall2) were administered by a 4 h intravenous infusion, to four patients and 35 healthy adult volunteers to evaluate toxicity. The dose-limiting toxicity (DLT) was observed at 74, 98, and 130 mg/m2, and included hepatotoxicity and diarrhea. Other reported adverse effects were mild and included nausea, abdominal distention, microscopic hematuria, elevated serum sodium, and skin rash [The toxicity and possible side effects of UA administration in humans has not been extensively studied. In one study by Wang et al. , single kin rash . OverallThe results from in vitro studies, reviewed herein, suggest that UA is active against prostate cancer cells inducing apoptosis and inhibiting cell cycle progression. The findings from a few animal studies indicate that UA could reduce tumor volume and increase survival in mice xenografted with prostate cancer cells. In addition, there are evidence from in vitro studies indicating the effects of UA against renal, bladder, and testicular cancers. A search of the literature did not reveal studies examining the combination of current chemotherapy agents such as docetaxel or paclitaxel and UA. Such studies will provide information about whether UA could act as a chemosensitizer. More animal studies as well as clinical trials should be performed in the future to determine the potential of UA to be used as an anti-cancer agent alone or in combination with other chemotherapy drugs to enhance their efficacy and potentially reduce the side effects experienced by patients. In addition, more animal and human clinical studies are required to examine the possible adverse effects and toxicity of oral administration of UA.


David Sacks and colleagues review the crosstalk between cell surface receptors and the IQGAP scaffold proteins. The scaffold protein IQGAP1 assembles multiprotein signaling complexes to influence biological functions. Cell surface receptors, particularly receptor tyrosine kinases and G-protein coupled receptors, are common IQGAP1 binding partners. Interactions with IQGAP1 modulate receptor expression, activation, and/or trafficking. Moreover, IQGAP1 couples extracellular stimuli to intracellular outcomes via scaffolding of signaling proteins downstream of activated receptors, including mitogen-activated protein kinases, constituents of the phosphatidylinositol 3-kinase pathway, small GTPases, and β-arrestins. Reciprocally, some receptors influence IQGAP1 expression, subcellular localization, binding properties, and post-translational modifications. Importantly, the receptor:IQGAP1 crosstalk has pathological implications ranging from diabetes and macular degeneration to carcinogenesis. Here, we describe the interactions of IQGAP1 with receptors, summarize how they modulate signaling, and discuss their contribution to pathology. We also address the emerging functions in receptor signaling of IQGAP2 and IQGAP3, the other human IQGAP proteins. Overall, this review emphasizes the fundamental roles of IQGAPs in coupling activated receptors to cellular homeostasis. IQGAPs are evolutionarily conserved scaffold proteins with a multidomain architecture, allowing interactions with numerous, diverse proteins. Like most vertebrates, humans have three IQGAP proteins: IQGAP1, IQGAP2, and IQGAP3 ; Box 1. Box 1The three IQGAP proteins2+-binding protein calmodulin, and the Rho GTPases Rac1 and Cdc42  share a similar multidomain composition, each containing a calponin homology domain (CHD), WW domain, IQ domain containing several IQ motifs, GTPase-activating protein (GAP)-related domain (GRD), and a RasGAP_C-terminal domain. IQGAPs are predominantly found in the cytosol, but also function at the plasma membrane to regulate receptor signaling and cell adhesion, at trafficking vesicles to coordinate endocytosis and exocytosis, and in the nucleus to influence transcription . All thrnd Cdc42 . Howevernd Cdc42 . IQGAP1 nd Cdc42 . In contnd Cdc42 . The mecBox 2IQGAP1 scaffolds the MAPK pathwayThe MAPK pathway transduces extracellular signals to cellular responses, such as differentiation, proliferation, and apoptosis. The pathway is initiated by the GTPase Ras that, upon activation by receptors, activates Raf kinase. Then, Raf activates by phosphorylation MEK, which in turn phosphorylates ERK. Activated ERKs translocate to the nucleus, where they regulate expression of target genes to mediate cellular outcomes . IQGAP1 Box 3IQGAP1 facilitates PI3K/Akt signaling2) to phosphatidylinositol-3,4,5-trisphosphate (PIP3). PIP3 binds the kinases PDK1 and Akt, allowing PDK1-mediated activation of Akt that catalyzes phosphorylation of various targets to promote cellular responses (2 from phosphatidylinositol (PI). Moreover, IQGAP1 binds PI3K, PDK1, and Akt to facilitate Akt activation  and G-protein coupled receptors (GPCRs), are common IQGAP1 interactors . IQGAP1 RTKs are characterized by intracellular tyrosine kinase activity. Activation upon binding of their cognate ligand most commonly induces RTK dimerization and autophosphorylation. Proteins are then recruited to the receptor primarily through Src homology 2 (SH2) and phosphotyrosine-binding (PTB) domains to initiate signaling . RTKs coThe erythroblastic leukemia viral oncogene homolog (ErbB) sub-family of RTKs comprises four structurally related members that mediate cell proliferation, differentiation, and migration . IQGAP1 EGFR: The EGFR:IQGAP1 complex was identified by mass spectrometry , predominantly expressed in vascular endothelial (VE) cells, stimulates cell proliferation and migration to promote angiogenesis. VEGF induces binding of IQGAP1 to VEGFR2, in complex with Rac1. IQGAP1 stabilizes GTP-bound Rac1, which activates the NAD(P)H oxidase Nox2, leading to the generation of reactive oxygen species  is an essential regulator of hematopoiesis and angiogenesis during development  controls embryo development and adult tissue homeostasis by regulating cell proliferation and differentiation . Activated IR recruits and phosphorylates insulin receptor substrate-1 (IRS-1), which recruits signaling proteins, including PI3K, which activates Akt to control GLUT4-mediated glucose uptake . The IQGsistance . Moreovesistance . CollectIn contrast to the other RTKs, IQGAP1 impairs Axl signaling. Axl is activated by Gas6 to control cell survival, proliferation, migration, and invasion . IQGAP1 MET is activated by the hepatocyte growth factor (HGF) to control cell growth, proliferation, survival, and motility, which drives embryogenesis and wound healing, but also tumorigenesis . IQGAP1 αβγ proteins  bind cytokines of the CXC family to mediate inflammatory and angiogenic functions . CXCR2, IQGAP1 also modulates signaling of CXCR4, which is activated by stromal-derived factor-1 SDF-1; . DepletiThe effects of opioids on pain are mediated by the µ-, δ-, κ-opioid, and nociception GPCRs. The μ-opioid receptor MOR1 is the main morphine receptor ; understThe δ-opioid receptor DOR1 is activated by the opioid peptide deltorphin. Analogous to CXCR4, IQGAP1 depletion impairs deltorphin-induced DOR1 intracellular trafficking and signaling to ERK . WhetherConsistent with its role in cell motility , IQGAP1 AR and ETBR. Activation of ET-1Rs by ET-1 in ovarian carcinoma cells increases IQGAP1 mRNA and protein levels. ET-1 also promotes interactions between IQGAP1 and β-arrestin1. This complex increases active RhoA and RhoC whereas, by recruiting RacGAP1, it inactivates Rac1 (AR/IQGAP1/β-arrestin1 positively correlates with poor prognosis in ovarian carcinoma patients (The peptide hormone endothelin-1 (ET-1) mediates vasoconstriction via two ET-1 receptors (ET-1Rs), ETtes Rac1 . By modutes Rac1 . High expatients , suggest2) enhances the DP1:IQGAP1 association and redistributes the two proteins to the perinuclear region. IQGAP1 knockdown reduces PGD2-induced DP1 internalization and ERK activation  interactome identified IQGAP1 . Moreovetivation . These dThe lysophosphatidic acid receptor 1 (LPA1), another lipid receptor, is overexpressed in breast carcinoma and increases metastasis . In breaLeucine-rich repeat-containing G protein–coupled receptors (LGRs) are activated by secreted R-spondins (RSPOs) to induce both canonical (Wnt/β-catenin) and non-canonical (β-catenin-independent) Wnt signaling . LGR4, oIQGAP1 also binds constitutively to LGR5 , anotherThe orphan G protein–coupled receptor 161 (GPR161) is overexpressed in breast cancer, where it promotes cell migration, proliferation, and invasion. IQGAP1 and β-arrestin2 co-immunoprecipitate with GPR161 from breast cancer cells. IQGAP1 knockdown attenuates GPR161-induced cell proliferation and migration by an unknown mechanism. Because GPR161 and IQGAP1 are both overexpressed in breast cancer , their c2+ signaling to promote cell proliferation. Stimulating Chinese hamster ovary cells transfected with M3-mAChRs with the muscarinic agonist carbachol promotes translocation of both Rac1 and IQGAP1 to cell junctions where they colocalize (M3-muscarinic acetylcholine receptors (M3-mAChRs) activate MAPK and Calocalize . M3-mAChlocalize , probabllocalize . IQGAP1 + tumor-infiltrating lymphocytes (TILs), impairs GPCR signaling by binding Gα , which is highly expressed in human CD8nding Gα . Mass sppitation . Rgs16 dterparts . The autα12, one of the four sub-families of Gα subunits of G proteins. Gα12 signaling is increased in primary nasopharyngeal carcinoma (NPC) cells  cells . Knockdonockdown . TogetheThough RTKs and GPCRs constitute the majority of transmembrane receptors with which IQGAP1 interacts, it also associates with receptor serine/threonine kinases (RSTKs), glutamate-gated ion channels (GICs), adhesion receptors, T cell receptors (TCRs), and receptor protein tyrosine phosphatases (RPTPs).RSTKs initiate signaling via their intracellular serine/threonine kinase activity . Transfo2+ flux mediated by activated NMDA stimulates binding of IQGAP1 to the microtubule-associated protein Lis1 to drive neuronal motility (Glutamate-gated ion channels (GICs) are cell surface receptors coupled to ion channels that coordinate synaptic transmission in the brain by mediating ion flux on binding the neurotransmitter glutamate . IQGAP1 motility . Togethe2+/calmodulin-dependent protein kinase II (CaMKII) are also in the complex . AJs are formed upon trans-dimerization of classic cadherin receptors on adjacent cells, each bound intracellularly to β- or γ-catenin and α-catenin linked to actin (The cadherin family are Cato actin . IQGAP1 to actin . This meto actin  and carcto actin . Bindingto actin , inhibitto actin , and stito actin . Similarto actin . Cells ato actin .IQGAP1 also interacts with VE-cadherin, which promotes VE-cadherin localization at sites of cell–cell contacts . Like E-Finally, IQGAP1 participates in cell adhesion via its interaction with the transmembrane glycoprotein CD44, the receptor for hyaluronan HA; . In ovar+ T cells exhibit increased proliferation and cytokine production following OX40 stimulation, illustrating that IQGAP1 restrains OX40 signaling (In immune cells, IQGAP1 negatively regulates TCR signaling. Upon TCR ligation, actin accumulation at immunological synapses is increased in cells depleted of IQGAP1. This upregulates TCR signaling and increases cytokine production . Moreoveignaling . Stimulaignaling , again signaling , implyinRPTPs modulate protein tyrosine phosphorylation using their intracellular phosphatase activity . Via thi1510 (1510 by MET induces the recruitment of SH2-containing proteins to IQGAP1 (1510 of IQGAP1 with non-phosphorylatable Ala impairs HGF/MET-stimulated Akt activation and cell migration, implying that this phosphorylation event influences IQGAP1 functions (1510 since c-Src overexpression increases phosphorylation of that residue (1441/1443. IQGAP1 with phosphorylated Ser1443 promotes EGFR activation, demonstrating positive feedback (2 (1441/1443 of IQGAP1 by an unknown mechanism, which stimulates binding and activation of Rac1 (IQGAP1 interacts with various RTKs, and some of them influence its tyrosine phosphorylation. MET is the only RTK documented to directly phosphorylate IQGAP1, and this occurs exclusively on Tyr1510 . Importao IQGAP1 . Replaceunctions . More inunctions ; possibl residue . Similarfeedback . EGF alsdback (2 , while Hdback (2 . Whether of Rac1 . The GPC of Rac1 . We suspParticipation of IQGAP2 and IQGAP3 in receptor signaling has recently emerged. To our knowledge, the only receptor to which IQGAP2 and IQGAP3 bind is the GPCR LGR4. While functions of the LGR4:IQGAP2 complex remain unidentified, IQGAP3 promotes RSPO/LGR4 signaling to Wnt/β-catenin, like IQGAP1 . SeveralIQGAP3, like IQGAP1, augments EGF-stimulated activation of EGFR and signaling to ERK, which has been suggested to enhance IQGAP3-driven tumorigenesis . In nerv1155 and Lys1230 decreases IQGAP1 binding to Cdc42 and Rac1 (1510 recruits selective SH2-containing proteins (The interactions of IQGAP proteins with receptors and signaling molecules must be tightly regulated in time and space to avoid disrupted and/or hyperactive signaling. IQGAP1 undergoes PTMs, some of which have been documented to modulate binding of signaling proteins. For example, ubiquitination of Lysand Rac1 , while pproteins . Interesproteins . This in2+/calmodulin and EGFR compete for binding to the IQGAP1 IQ domain (Other mechanisms could regulate IQGAP scaffolding. Because each IQGAP molecule binds numerous proteins, competition for binding could impair scaffolding. This concept has been experimentally demonstrated: CaQ domain . In contQ domain . IQGAP1 Q domain , which cQ domain . AlternaThe multidomain composition of IQGAP1 enables scaffolding of multiprotein complexes, comprising both receptors and signaling proteins. Thus, IQGAP1 functions as a central platform that couples cell surface receptor activation to intracellular responses. Although less characterized, analogous roles for IQGAP2 and IQGAP3 have recently emerged. The differential functions of the IQGAPs in receptor signaling confirm their non-redundancy and emphasize the importance of studying all three proteins. An important unanswered question is what mechanisms determine dynamic and selective assembly of IQGAP-mediated signalosomes. Further elucidation of the biochemical and cellular properties of IQGAPs, as well as structural characterization of IQGAP complexes, are required to enhance our understanding of regulation of scaffolding.Importantly, receptor:IQGAP complexes have been identified in several cancer cell lines, with tumorigenic implications. IQGAP-mediated receptor signaling also contributes to other diseases, like diabetes and macular degeneration. Pathogenic signaling may arise from overexpression of IQGAPs and/or receptors, as is commonly observed in neoplastic cells, increasing the number of receptor:IQGAP complexes and amplifying signaling. Alternatively, altered dynamic regulation of the complexes could disturb signaling, disrupting normal cell functions. Elucidating the molecular mechanisms by which receptor:IQGAP complexes contribute to disease could lead to the development of small molecule inhibitor therapeutics which could specifically and selectively target these interactions.


Hedgehog signaling plays pivotal roles in embryonic development, adult homeostasis and tumorigenesis. However, its engagement in the pituitary gland has been long underestimated although Hedgehog signaling and pituitary embryogenic development are closely linked. Thus, deregulation of this signaling pathway during pituitary development results in malformation of the gland. Research of the last years further implicates a regulatory role of Hedgehog signaling in the function of the adult pituitary, because its activity is also interlinked with homeostasis, hormone production, and most likely also formation of neoplasms of the gland. The fact that this pathway can be efficiently targeted by validated therapeutic strategies makes it a promising candidate for treating pituitary diseases. We here summarize the current knowledge about the importance of Hedgehog signaling during pituitary development and review recent data that highlight the impact of Hedgehog signaling in the healthy and the diseased adult pituitary gland. One mutant was almost entirely covered with denticles. Due to its ‘hedgehog-like’ appearance, the responsible gene was named hedgehog (hh). Simultaneously, the patch locus was identified, which later turned out to contain the gene encoding for the Hh receptor patched (Ptch)  and smoothened (smo)  . Subsequed (smo) , 3. TodaThe pituitary is one of the organs that depend on HH signaling –8. This 22.1In vertebrates, three secreted HH ligands exist: sonic HH (SHH), indian HH and desert HH. Of these secreted factors, SHH is the most important and best-characterized one. Major components of the HH signaling pathway in vertebrates comprise the secreted HH ligands (e.g. SHH), the HH receptor and 12-transmembrane protein PTCH1, the seven-transmembrane G protein-coupled receptor and interaction partner of PTCH1 SMO, the suppressor of fused homolog (SUFUH) as a negative regulator of HH signaling as well as the glioma-associated oncogene (GLI) family of transcription factors  (reviewed in –20) . Concomitantly, the HHN is modified with a cholesterol moiety at its C-terminus (HHNc) that allows tethering of HHNc to the outer cell membrane and further a palmitoyl group at its N-terminus (pHHNc) by HH acyltransferase (HHAT) , 22 Fi Figure reviewe). BOC, CThe released pHHNc molecules are then either captured by the HH interacting protein (HHIP), resulting in sequestration of the ligand, or by its receptor PTCH1  that forIn vertebrates, both HH signal reception and intracellular HH signal transmission require the primary or sensory cilium, which is a specialized antenna-like organelle that functions in signal sensing and transduction. The primary cilium is a singular protrusion from the cellular surface that can be found on almost every vertebrate cell. It consists of nine microtubule doublets that extend from the membrane-docked mature centriole  and form the microtubule-based cytoskeletal core of the cilium, the so called axoneme. The axoneme is surrounded by a ciliary membrane, which is a specialized and distinct compartment of the plasma membrane. Molecules and proteins essential for ciliogenesis, maintenance and signaling of the cilium are transported within the cilium bi-directionally along the microtubules by intraflagellar transport. With respect to HH signaling, initial signal reception, pathway activation and transcription factor processing are linked to the primary cilium (reviewed in ).R, GLI3R) that translocate to the nucleus and repress the transcription of HH signaling target genes . Since et genes . In contet genes –42.A/3A) and translocate into the nucleus, where they promote HH signaling target gene expression   .2.22+ influx that affects the cytoskeleton, angiogenesis, cellular differentiation, proliferation, apoptosis and migration. For further details, please refer to some excellent reviews on this topic .GLI1 transcription with the help of CRD-BP (coding region determinant-binding)  as well as PI3K/AKT signaling are able to stabilize GLI proteins and to stimulate their activity via mitogen-activated protein kinase (e.g. MEK or MAP2K) –52. Furtbinding)  or sulfabinding) , respectbinding) . Vice vetivation , GLI3R –-catenin . Additio-catenin , 61. GLI-catenin , 45).3sella turcica at the cranial base of the sphenoid bone. It is covered by a flat piece of dura mater, the diaphragma sellae. The pituitary borders laterally on the wall of the cavernous sinus and antero-inferiorly on the posterior wall of the sphenoid sinus. Antero-superiorly, it lies in close proximity to the optic chiasm . In most animals, the pars intermedia is distinct from the fleshy, glandular adenohypophysis and the neural composition of the neurohypophysis. However, in humans, the intermediate lobe is only few cell layers thick and normally either very small or entirely absent in adulthood. As a result, it is often considered as a part of the adenohypophysis  and the neurohypophysis (posterior lobe). The pituitary gland is connected with the superordinate hypothalamus via the infundibulum  and the hypophyseal portal system. The boundary between the adeno- and the neurohypophysis is called iewed in , 62, 63)iewed in Figure 23.1pars intermedia originates from an evagination of the oral ectoderm at the roof of the developing oral cavity  and thus is of epithelial origin. The neurohypophysis develops from descendants of the neural ectoderm, precisely from the ventral hypothalamic floor of the ventral diencephalon . Thll types   during murine embryogenesis. At this stage, a thickening of the ectoderm in the midline of the anterior neural ridge forms the hypophyseal placode that further develops into the RP between 9 to 10.5 dpc. By 10.5 dpc, when the definite RP has been formed, the neural ectoderm evaginates to form the infundibulum. The close and continuous contact of the neural ectoderm and the RP is essential for pituitary organogenesis. At 12.5 dpc, the pouch is completely separated from the underlying oral ectoderm. By 17 dpc, all parts of the murine pituitary have been developed. However, the lumen of the pouch persists as the pituitary cleft, separating the anterior and intermediate lobes in the mature gland  , paired-like homeobox proteins , LIM homeodomain transcription factors  and by SOX2 (SRY-box transcription factor 2), β-catenin, NOTCH and SHH. Secondly, the proliferation and invagination of RP cells, which are controlled by bone morphogenic proteins , fibroblast growth factors  and SHH. Thirdly, the lineage determination and cellular differentiation into endocrine cells, which is mediated by the stem cell marker PROP1 (homeobox protein prophet of PIT-1), PIT1 , GATA2 (GATA binding protein 2), NR5A1  and TPIT  , 64–66. 3.1.2Shh is expressed in the ventral diencephalon and the lateral oral ectoderm at 10 dpc in mice, before the formation of the RP . GLI1 expression is detectable throughout the developing hypothalamus and the RP , activates HH signaling in regions containing undifferentiated precursors/stem cells   .3.23.2.1The neurohypophysis is connected to the hypothalamus by the infundibulum. Magnocellular neurosecretory cells from the supraoptic and paraventricular nuclei of the hypothalamus project their axons to the neurohypophysis through the infundibulum Figure 2).Furthermore, the adenohypophysis is regulated by the hypothalamus via the hypophyseal portal system, which is a system of blood vessels. Neurosecretory axons in the hypothalamus terminate at the median eminence and release hypothalamic releasing and inhibitory hormones , growth hormone-releasing hormone, GHRH; gonadotropin-releasing hormone, GnRH; growth hormone-inhibiting hormone, GHIH; thyrotropin-releasing hormone, TRH). These releasing or inhibitory hormones reach the adenohypophysis via microcirculation and stimulate or inhibit the hormone secretion of their target endocrine cells, respectively  FiShh, Ptch1, Smo, Gli1, and Gpr161 in differentiated neurons of the adult mouse hypothalamus  and the pars intermedia  , adrenocorticotropic hormone (ACTH), prolactin (PRL), luteinizing hormone (LH) or follicle-stimulating hormone (FSH), and thyroid-stimulating hormone (TSH), respectively . Togethiewed in –125 (reviewed in ). Additiiewed in , 128). Tiewed in , 63) .Somatotrophs constitute about 30-40% of all adenohypophyseal cells. They produce and release GH that exerts its effects in almost all tissues. GH promotes linear growth by stimulating protein synthesis and transport of amino acid through the cells. In the liver, GH induces the production of the somatomedins IGF-1 (somatomedin C), IGF-2 (somatomedin A) and vitronectin , which stimulate cell division and cellular proliferation. For example, somatomedins enhance T-cell proliferation, increase the absorption of calcium from the intestine and reduce calcium loss through urination, stimulate hepatic glycogenolysis to raise levels of glucose in the blood, increase the growth of soft and skeletal tissues and increase the uptake of non-esterified fatty acids by the muscle. The release of GH from somatotrophs in the adenohypophysis is positively controlled by hypothalamic GHRH. Negative feedback loops that inhibit GH secretion are mediated by high circulating GH concentrations and by liver-born IGF-1 that stimulates hypothalamic GHIH secretion .pars intermedia produce and secrete α-MSH, β-MSH and γ-MSH. These proteins are derived from different regions of POMC and bind with different preferences to different melanocortin receptors. Circulating α-MSH induces the release of melanin pigment in skin melanocytes during human fetal life and upon sun exposure in adulthood. In lower vertebrates, such as fish or amphibians, Msh from the pars intermedia induces skin darkening in response to changes in background color, whereas Msh released from hypothalamic neurons controls feeding and energy expenditure (reviewed in (Corticotropic cells and melanotrophs express pro-opiomelanocortin (POMC) which is the precursor protein for ACTH and β-lipotropin (β-LPH) in corticotrophs and for melanocyte-stimulating hormones  in melanotrophs. In response to hypothalamic CRH stimulation, corticotrophs secrete ACTH that stimulates the adrenal cortex to produce cortisol. Via a negative feedback, cortisol inhibits the release of hypothalamic CRH and thus hypophyseal ACTH secretion. A functional role of β-LPH has not been proved so far. However, β-LPH is cleaved into β-endorphin and γ-LPH that is the precursor of β-MSH. In fact, corticotrophs produce and secrete β-endorphin acting as endogenous opioid neuropeptide that reduces stress and maintains homeostasis. Conversely, melanotrophs of the iewed in , 130).Lactotrophs comprise about 20% of all cells in the adenohypophysis. They produce and release PRL, which induces growth and development of the breast and maintains lactation in women. The production and secretion of PRL is primarily controlled by the hypothalamus that releases dopamine, which predominantly inhibits PRL release. Upon nipple stimulation of lactating women, sensory neurons are activated causing an inhibition of the dopamine release and thus an unopposed release of PRL. During pregnancy, the pituitary gland may increase by approximately 30% due to lactotroph hyperplasia .CGA gene) and beta subunits . The production and release of FSH and LH from the adenohypophysis are controlled by GnRH produced by the hypothalamus. Additionally, steroid sex hormones (e.g. estrogen) inhibit FSH and LH secretion via negative feedback loops  and TSH (see below) are glycoprotein hormones. All these hormones are heterodimers consisting of non-covalently associated alpha .Thyrotrophs compose around 5% of the adenohypophysis. They produce and release the glycoprotein hormone TSH that is controlled by the hypothalamic TRH. TSH functions as a stimulator of the thyroid gland to produce and release the hormones T3 and T4. In a negative feedback, T4 inhibits TSH secretion .+ FS cells subsequently regulate corticotrophs, which express ANXA1-associated G protein coupled receptors, via a paracrine mechanism  is detected simultaneously  express markers of active HH signaling. Therefore, HH signaling may be involved in pituitary homeostasis and/or tissue regeneration.Vila et al. were the first scientists, who evaluated the potential role of HH signaling in the adult human adenohypophysis , 14. Theaneously , 14. Thianeously , 13, 14.aneously , indicatry cells  and of Py glands . Additioy glands  who sequPomc transcription  are lost or severely reduced in these mice, suggesting a loss of neuronal differentiation due to interrupted HH signaling  and of the terminal differentiation markers POMC and β-TSH  during different zebrafish developmental stages revealed that HH signaling may also play a role even before invagination of the oral ectoderm and during early patterning of the gland  depletion results in reduced Fgf10 and increased Ptch1 expression in the infundibulum at 10.5 dpc. This goes along with hyperproliferation of the ventral diencephalon, hypoplasia of the adenohypophysis and lack of evagination of the infundibulum and thus of the neurohypophysis , holoprosencephaly (HPE), isolated or combined pituitary hormone deficiency (CPHD), congenital hypopituitarism without HPE, or pituitary stalk interruption syndrome (PSIS)   . Moreovee above) , 91 and cription .GLI3 gene (from nucleotides 1998-3481) that lead to an exclusive expression of the GLI3R .PTCH1    , 175. Thfunction , 175. CPfunction , 176. Hoe below) .PTCH1, PTCH2  that modulate HH signaling activity by regulating GLI3 processing (GLI3 (WFS1 (Wolframin ER transmembrane glycoprotein) mutations, which are found in 95% of Wolfram syndrome patients, lead to GLI1 degradation in in vitro analyses . HH-ass4.2.1Neoplastic lesions in the sellar region can be classified as PitNETs , hypotha+ PitNETs account for approximately 15% of PitNETs. Because these tumors secrete ACTH (corticotroph), which results in excess of ACTH, the phenotype of the patients resembles that of Cushing disease. PIT1+ PitNETs are subdivided into tumors, which release GH and/or CGA (somatotroph), PRL (lactotroph), TSH (thyrotroph) or GH together with PRL and/or TSH . Over 33% of resected PitNETs are SF1+ and secrete FSH and LH (gonadotroph) . Piiewed in . Togetheiewed in –190, 192iewed in Table 3.GNAS , which result in constitutive activation of cyclic AMP signaling (reviewed in (AIP (aryl hydrocarbon receptor interacting protein) mutations, whose role in tumorigenesis is still unclear , MAX (MYC associated factor X) or PRKAR1A  and ACTH-secreting tumors can harbor USP8 (ubiquitin specific peptidase 8), USP48, DICER1, MLH1 (MutL homolog 1) or MSH2 (MutS homolog 2) mutations. In addition, pituitary stalk hemangioblastomas can harbor VHL (Von Hippel-Lindau tumor suppressor) mutations . The tuiewed in ). Furtheiewed in )  (Tableiewed in ). Dependectively . GNAS musyndrome . 2% of asyndrome . 20% of  unknown . Patient unknown , 238. Th1B genes  (reviewe1B genes ). Moreov PitNETs , 234. Fulastomas . Finally PitNETs , 223.Hypothalamic tumors are subdivided into gangliocytoma, neurocytoma and pilocytic astrocytoma , whereas PPTs are categorized into pituicytoma, granular cell tumors (GCT), spindle cell oncocytoma (SCO), sellar ependymoma (SE). Other tumors of the sellar region are craniopharyngiomas, germinomas, suprasellar meningiomas, chordoma, chondroma, Rathke’s cleft cysts (RCC) and hamartoma. Except for PitNETs, tumors of the sellar region have different cellular origins such as the craniopharyngeal duct (craniopharyngiomas), or arachnoid cells (meningiomas) or are primordial germ cell remnants (germinomas). Nevertheless, pituicytomas, GCT, SCO and SE, that together account for only 0.5% of all sellar tumors, probably represent a spectrum of a unique neuropathological entity that most likely originate from pituicytes of the neurohypophysis . Whereas4.2.2in vitro. Additionally, active HH signaling induces USP48 expression via a positive feedback loop by GLI1 binding to the USP48 promoter , the auSHH/SHH, which strongly and significantly correlates with GLI1 mRNA expression levels. Remarkably, this is identical with classical HH-associated malignancies, which include the highly aggressive medulloblastoma and the benign basal cell carcinoma (see above). Since ACTH-, GH- and PRL-immunopositive PitNETs show the highest SHH and GLI1 levels, we propose that HH signaling activity may play a role in formation and/or maintenance of these pituitary tumor subtypes  is enriched for the expression of genes involved in HH signaling and expresses higher GLI1 and GLI2 levels  and CDKN2A (cyclin dependent kinase inhibitor 2A)   and tumo and NF1 . Interesuicytoma . Loss ofrminomas . Additiorminomas . These srminomas , is uprerminomas . Moreoverocytoma  and a strocytoma . Similarrocytoma . Active rocytoma , 253–256rocytoma . Similarutations , 259 thautations . This inutations Table 2.Together, these data suggest that HH signaling in APC and hypothalamic hamartomas has a rather tumor-suppressive function, whereas in hypothalamic endocrine tumors and PPTs it may have an oncogenic function. However, HH signaling activity in gangliocytoma, pilocytic astrocytoma , GCT, suprasellar meningioma or RCC has not been reported so far. It will be highly interesting to see whether HH signaling also plays a functional role in these tumor entities.5Research of the last twenty years has greatly improved our knowledge about the impact of HH signaling in the embryonic and adult pituitary. Animal experiments show that cells in the developing pituitary are competent to receive and respond to HH signaling, and that HH signaling promotes patterning, cell proliferation and terminal differentiation during pituitary organogenesis. In line with these observations, aberrant HH signaling activity is frequently found in human developmental disorders associated with pituitary malformations or hormone deficiencies, indicating that the HH signaling cascade plays an important role during pituitary development in humans as well. Very recent data further suggest that HH signaling is also active in adulthood and regulates pituitary homeostasis and hormone production. In addition, HH signaling seems to have a tumor-supportive role for PitNETs and PPTs. However, additional studies are needed to support these findings and to test whether HH signaling may be an intervention target for treatment of pituitary diseases./6-inhibitor abemaciclib in the treatment of meningioma (NCT02523014). They are also used in combination with gemcitabine, the CDK4/6-inhibitor ribociclib, the BRAF-inhibitor trametinib or the cytokine filgrastim for treatment of brain tumors  in children and young adults (NCT03434262). However, to date treatments of pituitary neoplasms with HH signaling inhibitors have been only reported in mouse models. For example, Ding et al. showed that inhibition of HH signaling by vismodegib blocks tumor growth of xenotransplanted sterol carrier protein 2-overexpressing GH3 cells  have been approved by the US Food and Drug Administration (FDA) and by the European Medicines Agency (EMA) for treating locally advanced basal cell carcinoma. Both drugs are currently tested in various clinical trials for other tumor entities either as a single agent or in combination with other anticancer drugs (reviewed in ). Thus, H3 cells . CurrentH3 cells ). AlthouH3 cells , 261. In NCT03434. HoweverH3 cells . For theH3 cells . Thus, tAll authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.