Evaluating the Activity of Smoothened Toward G Proteins Using [³5S]Guanosine 5'-(3-O-thio)triphosphate ([³5S]GTPγS).

The utilization of heterotrimeric G protein, and in particular those of the Gi, family, by Hedgehogs through Smoothened has become increasingly clear. We describe here a method for evaluating the activity of Smoothened toward G proteins in membranes derived from human embryonic kidney-293 (HEK293) cells. The assay relies on receptor-promoted exchange of GDP for [(35)S]GTPγS on the Gα subunit. The assay is best suited for analysis of the constitutive activity of Smoothened, inverse agonism superimposed on this activity, and neutral antagonism superimposed on inverse agonism. The assay would also be suitable for several other applications requiring a proximal, quantifiable readout of Smoothened activity.

Methods for Detection of Ptc1-Driven LacZ Expression in Adult Mouse Skin.

The Ptc-lacZ reporter mice are a highly utilized animal model for studying both normal tissue development and cancer. Identifying cell specific activation of Hedgehog (Hh) signaling is essential to understand the effects of this critical and complex signaling pathway. ß-gal detection in tissues can be difficult, with various staining procedures yielding differential results. Thus, detailed information on staining protocols is essential for determining the ideal method for a given study. Furthermore, immunohistochemical staining of X-Gal stained tissues can provide further insight into other key players in Hh signaling activation.

Trop-2 is up-regulated in invasive prostate cancer and displaces FAK from focal contacts.

In this study, we show that the transmembrane glycoprotein Trop-2 is up-regulated in human prostate cancer (PCa) with extracapsular extension (stages pT3/pT4) as compared to organ-confined (stage pT2) PCa. Consistent with this evidence, Trop-2 expression is found to be increased in metastatic prostate tumors of Transgenic Adenocarcinoma of Mouse Prostate mice and to strongly correlate with α5ß1 integrin levels. Using PCa cells, we show that Trop-2 specifically associates with the α5 integrin subunit, as binding to α3 is not observed, and that Trop-2 displaces focal adhesion kinase from focal contacts. In support of the role of Trop-2 as a promoter of PCa metastatic phenotype, we observe high expression of this molecule in exosomes purified from Trop-2-positive PCa cells. These vesicles are then found to promote migration of Trop-2-negative PCa cells on fibronectin, an α5ß1 integrin/focal adhesion kinase substrate, thus suggesting that the biological function of Trop-2 may be propagated to recipient cells. In summary, our findings show that Trop-2 promotes an α5ß1 integrin-dependent pro-metastatic signaling pathway in PCa cells and that the altered expression of Trop-2 may be utilized for early identification of capsule-invading PCa.

Crosstalk between Desmoglein 2 and Patched 1 accelerates chemical-induced skin tumorigenesis.

Aberrant activation of Hedgehog (Hh) signaling is causative of BCCs and has been associated with a fraction of SCCs. Desmoglein 2 (Dsg2) is an adhesion protein that is upregulated in many cancers and overexpression of Dsg2 in the epidermis renders mice more susceptible to squamous-derived neoplasia. Here we examined a potential crosstalk between Dsg2 and Hh signaling in skin tumorigenesis. Our findings show that Dsg2 modulates Gli1 expression, in vitro and in vivo. Ectopic expression of Dsg2 on Ptc1(+/lacZ) background enhanced epidermal proliferation and interfollicular activation of the Hh pathway. Furthermore, in response to DMBA/TPA, the Dsg2/Ptc1+/lacZ mice developed squamous lessons earlier than the WT, Ptc1(+/lacZ), and Inv-Dsg2 littermates. Additionally, DMBA/TPA induced BCC formation in all mice harboring the Ptc1(+/lacZ) gene and the presence of Dsg2 in Dsg2/Ptc1(+/lacZ) mice doubled the BCC tumor burden. Reporter analysis revealed activation of the Hh pathway in the BCC tumors. However, in the SCCs we observed Hh activity only in the underlying dermis of the tumors. Furthermore, Dsg2/Ptc1(+/lacZ) mice demonstrated enhanced MEK/Erk1/2 activation within the tumors and expression of Shh in the dermis. In summary, our results demonstrate that Dsg2 modulates Hh signaling, and this synergy may accelerate skin tumor development by different mechanisms.

Cell cycle- and cancer-associated gene networks activated by Dsg2: evidence of cystatin A deregulation and a potential role in cell-cell adhesion.

Cell-cell adhesion is paramount in providing and maintaining multicellular structure and signal transmission between cells. In the skin, disruption to desmosomal regulated intercellular connectivity may lead to disorders of keratinization and hyperproliferative disease including cancer. Recently we showed transgenic mice overexpressing desmoglein 2 (Dsg2) in the epidermis develop hyperplasia. Following microarray and gene network analysis, we demonstrate that Dsg2 caused a profound change in the transcriptome of keratinocytes in vivo and altered a number of genes important in epithelial dysplasia including: calcium-binding proteins (S100A8 and S100A9), members of the cyclin protein family, and the cysteine protease inhibitor cystatin A (CSTA). CSTA is deregulated in several skin cancers, including squamous cell carcinomas (SCC) and loss of function mutations lead to recessive skin fragility disorders. The microarray results were confirmed by qPCR, immunoblotting, and immunohistochemistry. CSTA was detected at high level throughout the newborn mouse epidermis but dramatically decreased with development and was detected predominantly in the differentiated layers. In human keratinocytes, knockdown of Dsg2 by siRNA or shRNA reduced CSTA expression. Furthermore, siRNA knockdown of CSTA resulted in cytoplasmic localization of Dsg2, perturbed cytokeratin 14 staining and reduced levels of desmoplakin in response to mechanical stretching. Both knockdown of either Dsg2 or CSTA induced loss of cell adhesion in a dispase-based assay and the effect was synergistic. Our findings here offer a novel pathway of CSTA regulation involving Dsg2 and a potential crosstalk between Dsg2 and CSTA that modulates cell adhesion. These results further support the recent human genetic findings that loss of function mutations in the CSTA gene result in skin fragility due to impaired cell-cell adhesion: autosomal-recessive exfoliative ichthyosis or acral peeling skin syndrome.

Patched-1 proapoptotic activity is downregulated by modification of K1413 by the E3 ubiquitin-protein ligase Itchy homolog.

The Hedgehog (Hh) receptor Patched-1 (PTCH1) opposes the activation of Gli transcription factors and induces cell death through a Gli-independent pathway. Here, we report that the C-terminal domain (CTD) of PTCH1 interacts with and is ubiquitylated on K1413 by the E3 ubiquitin-protein ligase Itchy homolog (Itch), a Nedd4 family member. Itch induces the ubiquitylation of K1413, the reduction of PTCH1 levels at the plasma membrane, and degradation, activating Gli transcriptional activity in the absence of Hh ligands. Silencing of Itch stabilizes PTCH1 and increases its level of retention at the plasma membrane. Itch is the preferential PTCH1 E3 ligase in the absence of Hh ligands, since of the other seven Nedd4 family members, only WW domain-containing protein 2 (WWP2) showed a minor redundant role. Like Itch depletion, mutation of the ubiquitylation site (K1314R) resulted in the accumulation of PTCH1 at the plasma membrane, prolongation of its half-life, and increased cell death by hyperactivation of caspase-9. Remarkably, Itch is the main determinant of PTCH1 stability under resting conditions but not in response to Sonic Hedgehog. In conclusion, our findings reveal that Itch is a key regulator of ligand-independent Gli activation and noncanonical Hh signaling by the governance of basal PTCH1 internalization and degradation.

Gi proteins mediate activation of the canonical hedgehog pathway in the myocardium.

During myocardial ischemia, upregulation of the hedgehog (Hh) pathway promotes neovascularization and increases cardiomyocyte survival. The canonical Hh pathway activates a transcriptional program through the Gli family of transcription factors by derepression of the seven-transmembrane protein smoothened (Smo). The mechanisms linking Smo to Gli are complex and, in some cell types, involve coupling of Smo to Gi proteins. In the present study, we investigated, for the first time, the transcriptional response of cardiomyocytes to sonic hedgehog (Shh) and the role of Gi protein utilization. Our results show that Shh strongly activates Gli1 expression by quantitative PCR in a Smo-dependent manner in neonatal rat ventricular cardiomyocytes. Microarray analysis of gene expression changes elicited by Shh and sensitive to a Smo inhibitor identified a small subset of 37 cardiomyocyte-specific genes regulated by Shh, including some in the PKA and purinergic signaling pathways. In addition, neonatal rat ventricular cardiomyocytes infected with an adenovirus encoding GiCT, a peptide that impairs receptor-Gi protein coupling, showed reduced activation of Hh targets. In vitro data were confirmed in transgenic mice with cardiomyocyte-inducible GiCT expression. Transgenic GiCT mice showed specific reduction of Gli1 expression in the heart under basal conditions and failed to upregulate the Hh pathway upon ischemia and reperfusion injury, unlike their littermate controls. This study characterizes, for the first time, the transcriptional response of cardiomyocytes to Shh and establishes a critical role for Smo coupling to Gi in Hh signaling in the normal and ischemic myocardium.

Smoothened is a fully competent activator of the heterotrimeric G protein G(i).

Smoothened (Smo) is a 7-transmembrane protein essential to the activation of Gli transcription factors (Gli) by hedgehog morphogens. The structure of Smo implies interactions with heterotrimeric G proteins, but the degree to which G proteins participate in the actions of hedgehogs remains controversial. We posit that the G(i) family of G proteins provides to hedgehogs the ability to expand well beyond the bounds of Gli. In this regard, we evaluate here the efficacy of Smo as it relates to the activation of G(i), by comparing Smo with the 5-hydroxytryptamine(1A) (5-HT(1A)) receptor, a quintessential G(i)-coupled receptor. We find that with use of [(35)S]guanosine 5'-(3-O-thio)triphosphate, first, with forms of G(i) endogenous to human embryonic kidney (HEK)-293 cells made to express epitope-tagged receptors and, second, with individual forms of Gα(i) fused to the C terminus of each receptor, Smo is equivalent to the 5-HT(1A) receptor in the assay as it relates to capacity to activate G(i). This finding is true regardless of subtype of G(i) (e.g., G(i2), G(o), and G(z)) tested. We also find that Smo endogenous to HEK-293 cells, ostensibly through inhibition of adenylyl cyclase, decreases intracellular levels of cAMP. The results indicate that Smo is a receptor that can engage not only Gli but also other more immediate effectors.

The Hedgehog signal transduction network.

Hedgehog (Hh) proteins regulate the development of a wide range of metazoan embryonic and adult structures, and disruption of Hh signaling pathways results in various human diseases. Here, we provide a comprehensive review of the signaling pathways regulated by Hh, consolidating data from a diverse array of organisms in a variety of scientific disciplines. Similar to the elucidation of many other signaling pathways, our knowledge of Hh signaling developed in a sequential manner centered on its earliest discoveries. Thus, our knowledge of Hh signaling has for the most part focused on elucidating the mechanism by which Hh regulates the Gli family of transcription factors, the so-called "canonical" Hh signaling pathway. However, in the past few years, numerous studies have shown that Hh proteins can also signal through Gli-independent mechanisms collectively referred to as "noncanonical" signaling pathways. Noncanonical Hh signaling is itself subdivided into two distinct signaling modules: (i) those not requiring Smoothened (Smo) and (ii) those downstream of Smo that do not require Gli transcription factors. Thus, Hh signaling is now proposed to occur through a variety of distinct context-dependent signaling modules that have the ability to crosstalk with one another to form an interacting, dynamic Hh signaling network.

Cholesterol and its derivatives in Sonic Hedgehog signaling and cancer.

The connection between the Hedgehog (HH) pathway and cholesterol has been recognized since the early days that shaped our current understanding of this unique pathway. Cholesterol and related lipids are intricately linked to HH signaling: from the role of cholesterol in HH biosynthesis to the modulation of HH signal reception and transduction by other sterols, passing by the phylogenetic relationships among many components of the HH pathway that resemble or contain lipid-binding domains. Here I review the connections between HH signaling, cholesterol and its derivatives and analyze the potential implications for HH-dependent cancers.

Noncanonical Hedgehog signaling.

The notion of noncanonical hedgehog (Hh) signaling in mammals has started to receive support from numerous observations. By noncanonical, we refer to all those cellular and tissue responses to any of the Hh isoforms that are independent of transcriptional changes mediated by the Gli family of transcription factors. In this chapter, we discuss the most recent findings that suggest that Patched1 can regulate cell proliferation and apoptosis independently of Smoothened (Smo) and Gli and the reports that Smo modulates actin cytoskeleton-dependent processes such as fibroblast migration, endothelial cell tubulogenesis, axonal extension, and neurite formation by diverse mechanisms that exclude any involvement of Gli-dependent transcription. We also acknowledge the existence of less stronger evidence of noncanonical signaling in Drosophila.

Sonic Hedgehog activates the GTPases Rac1 and RhoA in a Gli-independent manner through coupling of smoothened to Gi proteins.

The vertebrate Hedgehog (Hh) pathway has essential functions during development and tissue homeostasis in normal physiology, and its dysregulation is a common theme in cancer. The Hh ligands (Sonic Hh, Indian Hh, and Desert Hh) bind to the receptors Patched1 and Patched2, resulting in inhibition of their repressive effect on Smoothened (Smo). Smo is a seven-transmembrane protein, which was only recently shown to function as a G protein-coupled receptor (GPCR) with specificity toward the heterotrimeric guanine nucleotide-binding protein G(i). In addition to activating G(i), Smo signals through its C-terminal tail to inhibit Suppressor of Fused, resulting in stabilization and activation of the Gli family of transcription factors, which execute a transcriptional response to so-called "canonical Hh signaling." In this Presentation, we illustrate two outcomes of Hh signaling that are independent of Gli transcriptional activity and, thus, are defined as "noncanonical." One outcome is dependent on Smo coupling to G(i) proteins and exerts changes to the actin cytoskeleton through stimulation of the small guanosine triphosphatases (GTPases) RhoA and Rac1. These cytoskeletal changes promote migration in fibroblasts and tubulogenesis in endothelial cells. Signaling through the other noncanonical Hh pathway is independent of Smo and inhibits Patched1-induced cell death.

The alpha subunit of the G protein G13 regulates activity of one or more Gli transcription factors independently of smoothened.

Smoothened (Smo) is a seven-transmembrane (7-TM) receptor that is essential to most actions of the Hedgehog family of morphogens. We found previously that Smo couples to members of the G(i) family of heterotrimeric G proteins, which in some cases are integral although alone insufficient in the activation of Gli transcription factors through Hedgehog signaling. In response to a report that the G(12/13) family is relevant to Hedgehog signaling as well, we re-evaluated the coupling of Smo to one member of this family, G(13), and investigated the capacity of this and other G proteins to activate one or more of forms of Gli. We found no evidence that Smo couples directly to G(13). We found nonetheless that Gα(13) and to some extent Gα(q) and Gα(12) are able to effect activation of Gli(s). This capacity is realized in some cells, e.g. C3H10T1/2, MC3T3, and pancreatic cancer cells, but not all cells. The mechanism employed is distinct from that achieved through canonical Hedgehog signaling, as the activation does not involve autocrine signaling or in any other way require active Smo and does not necessarily involve enhanced transcription of Gli1. The activation by Gα(13) can be replicated through a G(q)/G(12/13)-coupled receptor, CCK(A), and is attenuated by inhibitors of p38 mitogen-activated protein kinase and Tec tyrosine kinases. We posit that G proteins, and perhaps G(13) in particular, provide access to Gli that is independent of Smo and that they thus establish a basis for control of at least some forms of Gli-mediated transcription apart from Hedgehogs.

Heterotrimeric Gi proteins link Hedgehog signaling to activation of Rho small GTPases to promote fibroblast migration.

Evidence supporting the functionality of Smoothened (SMO), an essential transducer in most pathways engaged by Hedgehog (Hh), as a G(i)-coupled receptor contrasts with the lack of an apparently consistent requirement for G(i) in Hh signal transduction. In the present study, we sought to evaluate the role of SMO-G(i) coupling in fibroblast migration induced by Sonic Hedgehog (Shh). Our results demonstrate an absolute requirement for G(i) in Shh-induced fibroblast migration. We found that Shh acutely stimulates the small Rho GTPases Rac1 and RhoA via SMO through a G(i) protein- and PI3K-dependent mechanism, and that these are required for cell migration. These responses were independent of transcription by Gli and of the C-terminal domain of SMO, as we show using a combination of molecular and genetic tools. Our findings provide a mechanistic model for fibroblast migration in response to Shh and underscore the role of SMO-G(i) coupling in non-canonical Hh signaling.

Hedgehog proteins activate pro-angiogenic responses in endothelial cells through non-canonical signaling pathways.

The Hedgehog (Hh) pathway orchestrates developmental and homeostatic angiogenesis. the three Hh isoforms--Sonic Hedgehog (Shh), Indian Hedgehog (Ihh) and Desert Hedgehog (Dhh)--signal through patched-1 (ptCH1) and Smoothened (SMo), to activate the Gli transcription factors with a characteristic rank of potency (Shh >> Ihh > Dhh). To dissect the mechanisms through which Hh proteins promote angiogenesis, we analyzed processes inherent to vessel formation in endothelial cells. We found that none of the Hh ligands were able to induce Gli-target genes in human umbilical vein (HUVeC) or human cardiac microvascular endothelial cells (HMVeC), suggesting that endothelial cells do not respond to Hh through the canonical pathway. However, our results show that the three Hh proteins promote endothelial cell tubulogenesis in 3D cultures in a SMo- and Gi protein-dependent manner. Consistent with the required cytoskeletal re-arrangement for tubulogenesis, Shh, Ihh and Dhh all stimulated the small GTPase RhoA and the formation of actin stress fibers. This effect, which was mediated by SMO, Gi proteins and Rac1, defines a new non-canonical Hh pathway. In addition to regulating the actin cytoskeleton, the Hh ligands promoted survival through inhibition of the pro-apoptotic effect of PTCH1 in a SMO-independent manner. Altogether, our results support the existence of Gli-independent Hh responses in endothelial cells that regulate tubulogenesis and apoptosis. The identification of novel non-canonical responses elicited by Hh proteins in endothelial cells highlights the complexity of the Hh signaling pathway and reveals striking differences in ligand strength for transcriptional and non-transcriptional responses

Purification and bioassay of hedgehog ligands for the study of cell death and survival.

The Hedgehog (Hh) family of secreted ligands-composed of Sonic Hedgehog (Shh), Indian Hedgehog (Ihh), and Desert Hedgehog (Dhh)-possesses many roles during embryonic development, adult homeostasis, and cancer. The specific functions of the Hh proteins are intertwined with their requirement as survival factors in Hh-responsive cells. However, studies designed to dissect the anti-apoptotic role of Hhs have been hindered by the lack of simple approaches to purify large quantities of recombinant ligands in the average laboratory setting because of the natural modifications of these proteins with palmitic acid and cholesterol. In this chapter, we provide a comprehensive protocol for the expression of Shh, Ihh, and Dhh in Escherichia coli as fusion proteins with calmodulin-binding peptide to allow easy and rapid purification. The ligands are engineered with a new N-terminus containing two isoleucine residues to provide an essential hydrophobic interphase for achieving high biologic activity. The protocol includes a detailed description of a method for determination of the specific activity of the generated proteins by use of a cell culture-based luciferase approach.

Early mitochondrial dysfunction in electron transfer activity and reactive oxygen species generation after cardiac arrest.

OBJECTIVE: Mitochondrial biology appears central to many conditions that progress to death but remains poorly characterized after cardiac arrest. Mitochondrial dysfunction in electron transfer and reactive oxygen species leakage during ischemia may lead to downstream events including mitochondrial protein oxidation, tyrosine nitrosylation, cytochrome c loss, and eventual death. We sought to better define early fixed alterations in these mitochondrial functions after whole animal cardiac arrest. METHODS: We used a murine model of 8 mins of untreated KCl-induced cardiac arrest followed by resuscitation and return of spontaneous circulation to study mitochondrial functions in four groups of animals: 1) after 8 min cardiac arrest (CA8) but no resuscitation, 2) 30 min postreturn of spontaneous circulation (R30), 3) 60 min postreturn of spontaneous circulation (R60), and in 4) shams. Heart mitochondria were immediately harvested, isolated, and stored at -80 degrees C for later spectrophotometric measurements of electron transfer activities and reactive oxygen species leakage using appropriate substrates and inhibitors. Mitochondrial cytochrome c content and tyrosine nitration were analyzed by Western blot and densitometry. RESULTS: A significant reactive oxygen species leakage from complex I was evident after just 8 min of cardiac arrest (CA8 group, p < .05), which was followed by a progressive reduction in complex I electron transfer activity (CA8 > R30 > R60). In contrast, complex II and II-III activities appeared more resistant to ischemia at the time points evaluated. Early changes in a approximately 50 kDa and approximately 25 kDa protein were observed in tyrosine nitration along with a loss of cytochrome c. CONCLUSIONS: A relatively "orderly" process of mitochondrial dysfunction progresses during ischemia and reperfusion. Changes in mitochondrial reactive oxygen species generation and electron transfer from complex I occur along with tyrosine nitrosylation and loss of cytochrome c; these may represent important new targets for future human therapies.

Pathways of signal transduction employed by vertebrate Hedgehogs.

Signalling by Hh (Hedgehog) proteins is among the most actively studied receptor-mediated phenomena relevant to development and post-embryonic homoeostatic events. The impact of signalling by the Hh proteins is profound, and work pertaining to the presentation of these proteins and the pathways engaged by them continues to yield unique insights into basic aspects of morphogenic signalling. We review here the mechanisms of signalling relevant to the actions of Hh proteins in vertebrates. We emphasize findings within the past several years on the recognition of, in particular, Sonic hedgehog by target cells, pathways of transduction employed by the seven-pass transmembrane protein Smoothened and end points of action, as manifest in the regulation of the Gli transcription factors. Topics of extended interest are those regarding the employment of heterotrimeric G-proteins and G-protein-coupled receptor kinases by Smoothened. We also address the pathways, insofar as known, linking Smoothened to the expression and stability of Gli1, Gli2 and Gli3. The mechanisms by which Hh proteins signal have few, if any, parallels. It is becoming clear in vertebrates, however, that several facets of signalling are shared in common with other venues of signalling. The challenge in understanding both the actions of Hh proteins and the overlapping forms of regulation will be in understanding, in molecular terms, both common and divergent signalling events.