Shh Cascade Research Articles

Development and Maintenance of Epidermal Stem Cells in Skin Adnexa.

The skin surface is modified by numerous appendages. These structures arise from epithelial stem cells (SCs) through the induction of epidermal placodes as a result of local signalling interplay with mesenchymal cells based on the Wnt–(Dkk4)–Eda–Shh cascade. Slight modifications of the cascade, with the participation of antagonistic signalling, decide whether multipotent epidermal SCs develop in interfollicular epidermis, scales, hair/feather follicles, nails or skin glands. This review describes the roles of epidermal SCs in the development of skin adnexa and interfollicular epidermis, as well as their maintenance. Each skin structure arises from distinct pools of epidermal SCs that are harboured in specific but different niches that control SC behaviour. Such relationships explain differences in marker and gene expression patterns between particular SC subsets. The activity of well-compartmentalized epidermal SCs is orchestrated with that of other skin cells not only along the hair cycle but also in the course of skin regeneration following injury. This review highlights several membrane markers, cytoplasmic proteins and transcription factors associated with epidermal SCs.

The Cytoskeletal Protein Zyxin Modulates the Expression of the Target Genes of the Shh Signaling Cascade in the Cells of the Neural Plate of Embryos of the Spur-Toed Frog Xenopus laevis

It has been shown earlier in a study of the role of the cytoskeletal protein zyxin in cell differentiation in the primordium of the central nervous system (CNS) of the spur-toed frog Xenopus laevis that zyxin interacts with three components of the signaling cascade of the secreted factor Sonic hedgehog (Shh): the transmembrane receptor Patched2 (Ptc2) and the transcription regulators Gli1 and Zic1. In the present work, the effect of zyxin knockdown on the expression of some key proteins of the Shh cascade has been studied. It has been shown using the RT-q PCR that the suppression of zyxin translation enhances the inhibitory effect of Shh on the expression of the target genes of this cascade such as Pax6, Irx3, and Dbx2 and affects the number of transcripts of several genes encoding the proteins that directly provide the functioning of the Shh cascade: Shh, Gli1, Ptc2, and Zic1. A hypothesis has been proposed that zyxin is involved in the regulation of the spatial marking of neural plate cells through the inhibitory effect on the Shh cascade.

Overexpression of laminin α4 facilitates proliferation and migration of fibroblasts in knee arthrofibrosis by targeting canonical Shh/Gli1 signaling

ABSTRACT Aim: Pathologic hyperplasia of fibroblast is responsible for the progression of intraarticular fibrosis. Laminin α4 (LAMA4), a subunit of laminin macromolecule family, was found to be overexpressed in various fibrotic tissues. However, the role of LAMA4 in knee arthrofibrosis remains elusive. Therefore, the aim of this study was to investigate the effect and mechanism of LAMA4 on fibroblast proliferation and migration. Materials and methods: Following knee surgery, LAMA4 expression was detected in intraarticular fibrous tissues in rabbits at week 2 and week 4, respectively. In lentivirus-mediated LAMA4-overexpressed fibroblasts, cellular proliferation was assessed by EdU labeling and cell cycle analysis, cellular migration was evaluated using Transwell assay, and the expressions of key components in Shh/Gli1 signaling were detected by qRT-PCR, western blot and immunofluorescence analysis. Additionally, canonical Shh cascade was further blocked in LAMA4-overexpressed fibroblasts by cyclopamine, and the changes in cellular proliferation and migration were investigated. Results: LAMA4 expression was positively correlated with the severity of knee arthrofibrosis. Functional studies demonstrated that LAMA4 overexpression facilitated proliferation, cell cycle progression and migration in fibroblasts. Mechanically, LAMA4 activated the canonical Shh/Gli1 signaling and promoted the nuclear translocation of Gli1 to upregulate expression of genes associated with cellular proliferation and migration. Intriguingly, blockage of Shh/Gli1 signaling with cyclopamine reversed the promoting effects of LAMA4 on proliferation and migration of fibroblasts. Conclusions: LAMA4 positively regulated cellular proliferation and migration in fibroblasts via activating the Shh/Gli1 signaling. LAMA4/Shh/Gli1 signaling axis might be a potential therapeutic target for the prevention of surgery-induced intraarticular fibrosis.

Ixazomib Modulates Bone Remodeling and Actives Sonic Hedgehog Pathways

Multiple myeloma (MM) is a clonal B-cell malignancy characterized by an accumulation of clonal plasma cells (PC) in the bone marrow (BM) leading to bone destruction and BM failure. Osteolytic bone disease is a common manifestation of MM that leads to progressive skeleton destruction and is the most severe cause of morbidity in MM patients. Pathogenetic mechanisms of MM bone destruction are closely linked to MM PC and osteoclasts (OCs) hyperactivity coupled with defective osteoblast (OB) function unable to counteract bone resorption. We recently demonstrated that the proteasome inhibitor (PI) Bortezomib, commonly used to treat MM, was capable to inhibit osteoclastic differentiation modulating chitinase family genes. In this work we investigated the effect of Ixazomib (IXA), a third generation PI, on osteoclastogenesis and osteogenic differentiation. Human monocytes were differentiated in OCs in presence of OC medium (supplemented with RANKL and M-CSF), and/or 10nM IXA. We observed that IXA was able to inhibit the expression of different OCs markers such as RANK, CTSK, TRAP, and MMP9 when added in OC medium in respect to OC medium alone (p<0.001). In addition, IXA treatment reduced CHIT1 enzymatic activity and downregulated CHIT1 and YKL40 (both mRNA and proteins). Immunofluorescence evaluation confirmed that IXA inhibited the mature OCs formation with five or more nuclei. Moreover, IXA was able to stimulate osteogenic differentiation of human mesenchymal stromal cells (MSCs). After 21 days of treatment, IXA alone or added to osteogenic medium increased the osteogenic markers genes (BMP2, RUNX2, Osteocalcin and Osteonectin). Immunofluorescence assay confirmed the increase of BMP2 after IXA treatment alone or in combination with osteogenic medium. Sonic Hedgehog (Shh) is one of the intricate signal transduction mechanisms that govern the precisely regulated developmental processes of multicellular organisms. Its signaling cascade plays an important role in bone homeostasis, and reducing Shh pathway protects against age-related bone loss. In this work we observed that IXA, but not Bortezomib, was able to bind the Smoothened (Smo) receptor which is the transducing molecule of the extracellular signal of Shh. Interestingly, using cellular thermal shift assay (CETSA), we demonstrated that IXA was able to bind directly Smo receptor activating the Shh cascade that leads to the nuclear translocation of Gli1 an effector of this pathway. Inhibiting Shh signaling by using an inhibitor of Smo activation (Cyclopamine), IXA-activated ostegenic differentiation-related genes were down-regulated. In conclusion, our data demonstrated that IXA regulates bone remodeling decreasing osteoclastogenesis and promoting osteogenic differentiation and therefore represents a good therapeutic option to improve the complex pathological condition of patients with MM. Disclosures Conticello: Celgene: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding. Palumbo:Amgen: Honoraria; Celgene: Honoraria; Hospira: Honoraria; Janssen: Honoraria; Novartis: Honoraria; Teva: Honoraria. Di Raimondo:Amgen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy; Celgene: Consultancy, Honoraria, Research Funding.

Abnormal Sonic hedgehog signaling in the lung of rats with esophageal atresia induced by adriamycin.

Abnormal lung development was recently described in the rat model of esophageal atresia and tracheoesophageal fistula (EA-TEF). Since in this condition the ventral-to-dorsal switch of Shh expression in the foregut is disturbed, the present study tested the hypothesis that this abnormal expression at the emergence of the tracheobronchial bud might be translated into the developing lung. Pregnant rats received either 1.75 mg/kg i.p. adriamycin or vehicle from E7 to E9. Three groups were studied: control and adriamycin-exposed with and without EA-TEF. Embryos were recovered and the lungs were harvested and processed for reverse transcription polymerase chain reaction and immunofluorescence analysis of the Shh signaling cascade. Shh signaling was downregulated at the late embryonic stage of lung development (E13) in embryos with EA-TEF. Throughout the subsequent stages of development, the expression of both Shh and its downstream components increased significantly and remained upregulated throughout gestation. Immunofluorescent localization was consistent with these findings. Defective Shh signaling environment in the foregut is present beyond the emergence of lung buds and probably impairs lung development. Later in gestation, lungs exhibited a remarkable ability to upregulate the Shh cascade, suggesting a compensatory response. These findings may be relevant to understand pulmonary disease suffered by children with EA-TEF.

Role of EST2/ETV5 as transcription factors in the regulation of SHH by FGF (541.3)

During limb development, fibroblast growth factors (FGFs) from the apical ectodermal ridge (AER) regulate the expression of Sonic hedgehog (SHH) in the underlying posterior distal mesoderm. The molecules or pathways involved in the regulation of SHH by FGF are unknown. A recent study suggests that ETS and ETV, members of the ETS transcription factor family, are intermediate regulators in the Fgf to Shh cascade and directly interact with the limb‐specific Shh regulatory region in the murine limb model.To determine whether this was a conserved function of ETS/ETV transcription factors, we used the chick model and examined ETS2/ETV5 expression by in situ hybridization following ectopic application of FGF in the posterior limb bud, an assay known to induce SHH.ETV5 is expressed in the distal mesoderm sub‐adjacent to the AER and overlaps the SHH expression domain. ETS2 expression is most prominent in the developing chick zeugopod and appears to correlate with the condensing radius and ulna. The posterior expression of ETS2 is more robust than its anterior expression. Both factors show increased expression around ectopically applied FGF2‐laden beads at 24 hours after bead implantation.These data demonstrate expression patterns that spatially complement and overlap the SHH expression domain. Furthermore, both factors are upregulated by ectopic FGF prior to or coincident with SHH upregulation. These data support a role for ETS transcription factors in the regulation of SHH by FGF.

Guggulsterone sensitizes glioblastoma cells to Sonic hedgehog inhibitor SANT-1 induced apoptosis in a Ras/NFκB dependent manner

Since Shh pathway effector, Gli1, is overexpressed in gliomas, we investigated the effect of novel Shh inhibitor SANT-1 on glioma cell viability. Though SANT-1 failed to induce apoptosis, it reduced proliferation of glioma stem-like cells. Apart from canonical Shh cascade, Gli1 is also induced by non-canonical pathways including NFκB. Therefore, a combinatorial strategy with Ras/NFκB inhibitor, Guggulsterone, was employed to enhance effectiveness of SANT-1. Guggulsterone inhibited Ras and NFκB activity and sensitized cells to SANT-1 induced apoptosis via intrinsic apoptotic mechanism. Inhibition of either Ras or NFκB activity was sufficient to sensitize cells to SANT-1. Guggulsterone induced ERK activation also contributed to Caspase-9 activation. Since SANT-1 and Guggulsterone differentially target stem-like and non-stem glioma cells respectively, this combination warrants investigation as an effective anti-glioma therapy.

Blockade of the sonic hedgehog signalling pathway inhibits choroidal neovascularization in a laser-induced rat model

Sonic hedgehog (Shh) signaling has recently been shown to be involved in the pathological angiogenesis in response to tissue hypoxia and ischemic injury. Hypoxia/ischemia is considered to play an important role in the development of choroidal neovascularization (CNV). This study was aimed to examine the effect of blockade of the Shh signaling pathway on CNV and the underlying mechanism. A total of 64 male Brown-Norway (BN) rats were used in this study. One eye of each rat underwent laser photocoagulation. The other eye served as normal control. After the laser treatment, the 64 rats were divided into four groups (n=16 in each group): Blank control group, in which no intravitreal administration was given; cyclopamine group, recombinant Shh N-terminals protein (rShh) group and phosphate-buffered saline (PBS) group, in which cyclopamine (a Shh inhibitor), rShh (a Shh activator) and PBS were intravitreally injected into the laser-treated eyes respectively every other day for a total of four intravitreal injections immediately after the laser treatment. Fourteen days after the intravitreal administration, the changes of CNV-related variables, including positive CNV lesion percentage, CNV membrane area and CNV membrane thickness, were evaluated by fluorescein angiography, indocyanine green angiography and pathological examinations. The mRNA and protein expression of PTCH1, Gli1, HIF-1(α), VEGF and DLL4 in each group on 14 days of CNV model was detected by real-time quantitative PCR and western blot analysis, and the relationship between the Shh cascade and the HIF-1(α)-VEGF-DLL4 cascade in CNV was analyzed. The results showed that the CNV membrane area and the CNV membrane thickness were decreased by 62.5% and 41.9% in the cyclopamine group and increased by 85.7% and 64.3% in the rShh group in comparison to those in the blank control group (P<0.01 for each). There was no significant difference in the CNV membrane area and thickness between the blank control group and PBS group (P=0.102 and P=0.063, respectively). Real-time quantitative PCR revealed a 5.23-, 4.14-, 2.97-, 2.78- and 2.39-fold up-regulation of the mRNA expression of PTCH1, Gli1, HIF-1(α), VEGF and DLL4 genes in the laser-treated eyes compared with the normal control eyes in the control group. In the cyclopamine group, the mRNA and protein expression of Gli1, HIF-1(α), VEGF and DLL4 was significantly down-regulated (P<0.05 for each) while the expression of PTCH1 showed no significant changes at the mRNA (P=0.293) and protein level (P=0.304). The mRNA expression and protein expression (P=0.001 and P=0.021, respectively) of PTCH1, Gli1, HIF-1(α), VEGF and DLL4 was significantly increased in the rShh group when compared with the control group. The expression level of these genes was related to the severity of the CNV. It was concluded that intravitreal administration of cyclopamine can effectively inhibit the formation of laser-induced experimental CNV by down-regulating the expression of the HIF-1(α)-VEGF-DLL4 cascade in CNV. The Shh signaling pathway as an upstream signaling pathway of HIF-1(α)-VEGF-DLL4 cascade is implicated in the development of experimental CNV.

Sonic hedgehog gene expression in nitrofen induced hypoplastic lungs in mice

The pathogenesis of congenital diaphragmatic hernia (CDH) is unknown. The sonic hedgehog (Shh) cascade is crucial for the patterning of the early respiratory system in mice. Optical Projection Tomography (OPT) is a new tool for 3D imaging small biological specimens that enables us to visualise both the anatomy of developing organs and gene expression localised in the context of normal or abnormal anatomy. We visualised Shh gene expression patterns in the nitrofen treated and control mouse lung buds at early stages of lung development. Time-mated CD1 female mice (n = 5) received oral administration of 100 mg of the herbicide 2,4-dichlorophenyl-p-nitrophenylether (nitrofen) (WAKO Chemical, Osaka, Japan) in 1 ml of olive oil or olive oil alone at 7 days of gestation. Embryos were harvested on gestation days 9-12, and stained following whole mount in situ hybridisation with labelled RNA probes to detect Shh transcripts at each stage. Embryos were scanned by OPT to obtain 3D representations of gene expression domains in the context of the changing morphology of the embryo. OPT analysis of Shh transcript distribution clearly revealed gene expression in both groups. In treated embryos, there were no significant changes in Shh transcript distribution in lung buds in comparison with control embryos. Although altered Shh expression in the hypoplastic lung has been reported in late gestation, the present study did not reveal any significant alterations in pulmonary Shh spatial transcript distribution or gene expression level during the early gestation in nitrofen CDH model. It would be of great interest in future studies to use OPT approach to investigate pulmonary expression of Shh and other regulatory genes both during early and late stages of lung development in order to provide new insights into the pathogenesis of pulmonary hypoplasia.

Monoaminergic regulation of Sonic hedgehog signaling cascade expression in the adult rat hippocampus

Monoamines are implicated in the modulation of adult hippocampal neurogenesis in depression models and following chronic antidepressant treatment. Given the key role of Sonic hedgehog (Shh) in adult neurogenesis, we examined whether monoaminergic perturbations regulate the expression of Shh or its co-receptors Smoothened (Smo) and Patched (Ptc). Combined depletion of both serotonin and norepinephrine with para-chlorophenylalanine (PCPA) resulted in a significant decrease in Smo and Ptc mRNA within the dentate gyrus subfield of the hippocampus. However, selective depletion of serotonin, using the serotonergic neurotoxin 5,7-dihyrdroxytryptamine (5,7-DHT), or norepinephrine, using the noradrenergic neurotoxin DSP-4, did not alter expression of Shh and its co-receptors, Smo and Ptc. Acute treatment with the monoamine releasing agent, para-chloroamphetamine (PCA) significantly upregulated Smo mRNA within the dentate gyrus. However, acute or chronic treatment with pharmacological antidepressants that modulate monoaminergic neurotransmission did not regulate Shh cascade expression. These results indicate that robust changes in monoamine levels can regulate the expression of the Shh signaling cascade in the adult rodent brain.

PS13 DEVELOPMENT OF THE URINARY BLADDER IN ETU‐EXPOSED FETAL RATS IS AFFECTED BY DOWNREGULATION OF SHH AND ITS TARGET GENESCAGES

Objective The cloaca develops into the urinary bladder and distal part of the hindgut. Abnormalities of cloacal development result in a variety of anorectal and urogenital malformations. This study evaluated the formation of the urinary bladder in ETU exposed fetal rats in relation to shh and its target genes.Methodology Embryos of pregnant SD females administered 1% ETU (125 mg/kg) on the tenth day of gestation (gD10) were collected from gD12‐16 and gD21. Cloacal development was viewed with serial histology followed by qualitative and quantitative gene expression studies of shh and target genes to elucidate the expression pattern of these genes during each time point.Results ETU exposed fetuses have incomplete separation of the cloaca leading to a variety of anorectal and urinary abnormalities eg rectourethral fistula. Shh and downstream genes are expressed during urinary bladder development in fetal rats. Relative quantification showed that expression of shh, BMP4 and Hox genes are reduced in ETU‐exposed fetal rats during the process of cloacal separation and development of the urinary bladder.Conclusion This study demonstrates that ETU‐exposure in fetal rats produces abnormal cloacal development, including urinary fistulae. Shh cascade is active during the process of cloacal separation and urinary bladder development and down regulation of shh, BMP4 and Hox genes during this process may contribute to the abnormal development of urinary bladder and anorectum.

Sonic hedgehog, BMP4, and Hox genes in the development of anorectal malformations in Ethylenethiourea-exposed fetal rats

Background shh signaling pathway has been shown to be involved in the morphogenesis of many organ systems. In this study, we investigated the expression of shh and its targets, BMP4 and Hox genes, in the development of anorectal malformations in Ethylenethiourea (ETU)-exposed embryos. Methods We used ETU murine model of the vertebral, anal, cardiac, tracheoesophageal, renal, and limb association. Ethylenethiourea 1% (125 mg/kg) was given to the pregnant females via gavage feeding on gestational day (gD) 10 and saline to control animals. Embryos were collected at gD12 to gD16 and gD21; hindguts were dissected and snap frozen. Highly purified RNA was isolated, and expression of shh, BMP4, Hoxa13, and Hoxd13 genes was confirmed with RT-PCR. Relative quantitative expression of shh and target genes at each time point was done with SYBR Green I qPCR. Normalized gene of interest expression was calculated by geNorm, and data analysis was done with 2-tail Student t test. Results shh, BMP4, Hoxa13, and Hoxd13 transcripts were detected in all samples, confirming that shh cascade is active during the process of hindgut development in fetal rats. Relative quantitation demonstrated that shh cascade expression shows time-dependent changes in the developing hindgut. Conclusion This study shows that ETU disturbs the expression of shh signaling pathway during the development of hindgut. We provide evidence that shh plays a pivotal role in the hindgut morphogenesis, and its misexpression affect the expression of targets, BMP4 and Hox genes.

Sonic hedgehog expression in the development of hindgut in ETU-exposed fetal rats

Sonic hedgehog (Shh) has been shown to be involved in the morphogenesis of many organ systems including the notochord, floor plate and limbs, as well as in the development of the left-right axis in vertebrates. Recent evidence suggests the Shh cascade plays a crucial role in the development of the foregut and hindgut. We have previously shown that prenatal exposure of fetal rats to ethylenethiourea (ETU) induces hindgut malformations and other abnormalities of the VACTERL association. The aim of this study was to determine the pattern of expression of Shh and its downstream genes during hindgut development in ETU-exposed embryos with anorectal malformations (ARMs). Pregnant Sprague-Dawley rats were mated together overnight and a positive vaginal plug was marked as gD0. On gD10, 1% ETU (125 mg/kg) was given to the experimental group and controls received the same volume of saline. Embryos were collected from both groups at gD12-16. The developing hindgut of each embryo was dissected under magnification and snap frozen. Highly purified RNA was isolated from each hindgut and first strand cDNA was prepared with appropriate negative controls. Reverse transcriptase (RT) polymerase chain reaction (PCR) was done to determine the transcripts of Shh in each sample and quantitative real-time PCR was carried out to show relative quantitative expression of Shh at each time point. Shh was detected in all samples confirming that Shh is active during the process of hindgut development in fetal rats. Relative quantitation demonstrated that Shh expression shows time-dependent changes in the developing hindgut of ETU-exposed rat embryos, and when results were compared with control samples, there was significant decrease in expression on gD14 and 15, when the cloaca normally separates into the rectum and urethra occurs in the rat fetus. The misregulated expression of Shh in the hindgut of ETU-exposed rat embryos suggests that ETU may interfere with Shh signalling. Downregulation at the time of cloacal separation into rectum and urethra indicates that Shh plays a crucial role in the development of hindgut.

Sonic hedgehog cascade is required for penile postnatal morphogenesis, differentiation, and adult homeostasis.

The penis is unique in that it undergoes morphogenesis and differentiation primarily in the postnatal period. For complex structures such as the penis to be made from undifferentiated precursor cells, proliferation, differentiation, and patterning are required. This process involves coordinated activity of multiple signals. Sonic hedgehog (Shh) forms part of a regulatory cascade that is essential for growth and morphogenesis of many tissues. It is hypothesized that the penis utilizes regulatory mechanisms similar to those of the limb and accessory sex organs to pattern penile postnatal morphogenesis and differentiation and that the Shh cascade is critical to this process. To test this hypothesis, Shh, BMP-4, Ptc, and Hoxa-10 localization and function were examined in Sprague-Dawley rat penes by means of quantitative reverse transcription polymerase chain reaction, in situ hybridization, immunohistochemistry, and Western blotting. These genes were expressed in the penis during postnatal morphogenesis in a spatially and temporally restricted manner in adjacent layers of the corpora cavernosal sinusoids. The function of Shh and BMP-4 is to establish and maintain corpora cavernosal sinusoids. The data suggest that Ptc and Hoxa-10 are also important in penile morphogenesis. The continuing function of Shh and targets of its signaling in maintaining penile homeostasis in the adult is significant because disruption of Shh signaling affects erectile function. This is the first report that demonstrates the significant role that Shh plays in establishing and maintaining penile homeostasis and how this relates to erectile function. These studies provide valuable insight that may be applied to improve treatment options for erectile dysfunction.