Regulation of cavernous nerve injury-induced apoptosis by sonic hedgehog

Thirty to eighty-seven percent of patients treated by radical prostatectomy experience erectile dysfunction (ED). The reduced efficacy of treatments in this population makes novel therapeutic approaches to treat ED essential. We propose that abundant apoptosis observed in penile smooth muscle when the cavernous nerve (CN) is cut (mimicking the neural injury which can result from prostatectomy) is a major contributing factor to ED development. We hypothesize that decreased Sonic hedgehog (SHH) signaling is a cause of ED in neurological models of impotence by increasing apoptosis in penile smooth muscle. We examined this hypothesis in a bilateral CN injury model of ED. We found that the active form of SHH protein was significantly decreased 1.2-fold following CN injury, that SHH inhibition causes a 12-fold increase in smooth muscle apoptosis in the penis, and that SHH treatment at the time of CN injury was able to decrease CN injury-induced apoptosis (1-3-fold) in a dose-dependent manner. These results show that SHH stabilizes the alterations of the corpora cavernosal smooth muscle following nerve injury.     

Cooperation of sonic hedgehog enhancers in midline expression

In zebrafish, as in other vertebrates, the secreted signalling molecule Sonic hedgehog (Shh) is expressed in organiser regions such as the embryonic midline and the zona limitans intrathalamica (zli). To investigate the regulatory mechanisms underlying the pattern of shh expression, we carried out a systematic analysis of the intronic regulatory sequences of zebrafish shh using stable transgenesis. Deletion analysis identified the modules responsible for expression in the embryonic shield, the hypothalamus and the zli and confirmed the activities of previously identified notochord and floor plate enhancers. We detected a strong synergism between regulatory regions. The degree of synergy varied over time in the hypothalamus suggesting different mechanisms for initiation and maintenance of expression. Our data show that the pattern of shh expression in the embryonic central nervous system involves an intricate crosstalk of at least 4 different regulatory regions. When compared to the enhancer activities of the mouse Shh gene, we observed a remarkable divergence of function of structurally conserved enhancer sequences. The activating region ar-C (61% identical to SFPE2 in mouse Shh), for example, mediates floor plate expression in the mouse embryo while it directs expression in the forebrain and the notochord and only weakly in the floor plate in the zebrafish embryo. This raises doubts on the predictive power of phylogenetic footprinting and indicates a stunning divergence of function of structurally conserved regulatory modules during vertebrate evolution.     

Neural influences on sonic hedgehog and apoptosis in the rat penis

The role of sonic hedgehog (SHH) in maintaining corpora cavernosal morphology in the adult penis has been established; however, the mechanism of how SHH itself is regulated remains unclear. Since decreased SHH protein is a cause of smooth muscle apoptosis and erectile dysfunction (ED) in the penis, and SHH treatment can suppress cavernous nerve (CN) injury-induced apoptosis, the question of how SHH signaling is regulated is significant. It is likely that neural input is involved in this process since two models of neuropathy-induced ED exhibit decreased SHH protein and increased apoptosis in the penis. We propose the hypothesis that SHH abundance in the corpora cavernosa is regulated by SHH signaling in the pelvic ganglia, neural activity, or neural transport of a trophic factor from the pelvic ganglia to the corpora. We have examined each of these potential mechanisms. SHH inhibition in the penis shows a 12-fold increase in smooth muscle apoptosis. SHH inhibition in the pelvic ganglia causes significantly increased apoptosis (1.3-fold) and decreased SHH protein (1.1-fold) in the corpora cavernosa. SHH protein is not transported by the CN. Colchicine treatment of the CN resulted in significantly increased smooth muscle apoptosis (1.2-fold) and decreased SHH protein (1.3-fold) in the penis. Lidocaine treatment of the CN caused a similar increase in apoptosis (1.6-fold) and decrease in SHH protein (1.3-fold) in the penis. These results show that neural activity and a trophic factor from the pelvic ganglia/CN are necessary to regulate SHH protein and smooth muscle abundance in the penis.     

Discovery of sonic hedgehog expression in postnatal growth plate chondrocytes: differential regulation of sonic and Indian hedgehog by retinoic acid

Sonic hedgehog (Shh) is a key signal protein in early embryological patterning of limb bud development. Its analog, Indian hedgehog (Ihh), primarily expressed during early cartilage development in prehypertrophic chondrocytes, regulates proliferation and suppresses terminal differentiation of postnatal growth plate (GP) chondrocytes. We report here for the first time that both Shh and Ihh mRNA are expressed in the GP of rapidly growing 6-week-old broiler-strain chickens. They are also expressed in other tissues such as articular chondrocytes, kidney, and bone. In situ hybridization and RT-PCR analyses reveal Shh in all zones of the GP, with peak expression in late hypertrophy. Using primary cultures of GP chondrocytes in serum-containing medium, we followed the patterns of Shh and Ihh mRNA expression as the cultures matured and mineralized. We find a cyclical expression of both hedgehog genes during the early period of culture development between day 10 and 14; when one is elevated, the other tended to be suppressed, suggesting that the two hedgehogs may play complementary roles during GP development. Retinoic acid (RA), a powerful modulator of gene expression in cell differentiation, stimulates GP chondrocytes toward terminal differentiation, enhancing mineral formation. We find that RA strongly suppresses Ihh, but enhances expression of Shh in this system. While Ihh suppresses maturation of GP chondrocytes to hypertrophy, we hypothesize that Shh acts to push these cells toward hypertrophy.     

Adenohypophysis formation in the zebrafish and its dependence on sonic hedgehog

Formation of the adenohypophysis in mammalian embryos occurs via an invagination of the oral ectoderm to form Rathke's pouch, which becomes exposed to opposing dorsoventral gradients of signaling proteins governing specification of the different hormone-producing pituitary cell types. One signal promoting pituitary cell proliferation and differentiation to ventral cell types is Sonic hedgehog (Shh) from the oral ectoderm. To study pituitary formation and patterning in zebrafish, we cloned four cDNAs encoding different pituitary hormones, prolactin (prl), proopiomelancortin (pomc), thyroid stimulating hormone (tsh), and growth hormone (gh), and analyzed their expression patterns relative to that of the pituitary marker lim3. prl and pomc start to be expressed at the lateral edges of the lim3 expression domain, before pituitary cells move into the head. This indicates that patterning of the pituitary anlage and terminal differentiation of pituitary cells starts while cells are still organized in a placodal fashion at the anterior edge of the developing brain. Following the expression pattern of prl and pomc during development, we show that no pituitary-specific invagination equivalent to Rathke's pouch formation takes place. Rather, pituitary cells move inwards together with stomodeal cells during oral cavity formation, with medial cells of the placode ending up posterior and lateral cells ending up anterior, resulting in an anterior-posterior, rather than a dorsoventral, patterning of the adenohypophysis. Carrying out loss- and gain-of-function experiments, we show that Shh from the ventral diencephalon plays a crucial role during induction, patterning, and growth of the zebrafish adenohypophysis. The phenotypes are very similar to those obtained upon pituitary-specific inactivation or overexpression of Shh in mouse embryo, suggesting that the role of Shh during pituitary development has been largely conserved between fish and mice, despite the different modes of pituitary formation in the two vertebrate classes.     

Localization of Indian hedgehog and PTH/PTHrP receptor expression in relation to chondrocyte proliferation during mouse bone development

We have developed a useful approach to examine the pattern of gene expression in comparison to cell proliferation, using double in situ hybridization and immunofluorescence. Using this system, we examined the expression of Indian hedgehog (Ihh) and PTH/PTHrP receptor (PPR) mRNA in relation to chondrocyte proliferation during embryonic mouse bone development. Both genes are expressed strongly in prehypertrophic and early hypertrophic chondrocytes, and there is a strong correlation between upregulation of both Ihh and PPR expression and chondrocyte cell cycle arrest. At embryonic day (E14.5), PPR mRNA upregulation begins in the columnar chondrocytes just prior to cell cycle exit, but at later time points expression is only observed in the postproliferative region. In contrast, Ihh mRNA expression overlaps slightly with the region of columnar proliferating chondrocytes at all stages. This study provides further evidence that in the developing growth plate, cell cycle exit and upregulation of Ihh and PPR mRNA expression are coupled.     

Indian hedgehog is an essential component of mechanotransduction complex to stimulate chondrocyte proliferation

Indian hedgehog (Ihh), a member of the vertebrate hedgehog morphogen family, is a key signaling molecule that controls chondrocyte proliferation and differentiation. In this study, we show a novel function of Ihh. Namely, it acts as an essential mediator of mechanotransduction in cartilage. Cyclic mechanical stress greatly induces the expression of Ihh by chondrocytes. This induction is abolished by gadolinium, an inhibitor of stretch-activated channels. This suggests that the IHH gene is mechanoresponsive. The mechano-induction of Ihh is essential for stimulating chondrocyte proliferation by mechanical loading. The presence of an Ihh functional blocking antibody during loading completely abolishes the stimulatory effect of mechanical load on proliferation. Furthermore, Ihh mediates the mechanotransduction process in a bone morphogenic protein (BMP)-dependent and parathyroid hormone-related peptide-independent manner. BMP 2/4 are up-regulated by mechanical stress through the induction of Ihh, and BMP antagonist noggin inhibits mechanical stimulation of chondrocyte proliferation. This suggests BMP lies downstream of Ihh in mechanotransduction pathway. Our data suggest that Ihh may transduce mechanical signals during cartilage growth and repair processes.     

Stathmin inhibits proliferation and differentiation of dental pulp stem cells via sonic hedgehog/Gli

The mineralization of dental pulp stem cells is an important factor in the tissue engineering of teeth, but the mechanism is not yet obvious. This study aimed to identify the effect of Stathmin on the proliferation and osteogenic/odontoblastic differentiation of human dental pulp stem cells (hDPSCs) and to explore whether the Shh signalling pathway was involved in this regulation. First, Stathmin was expressed in the cytoplasm and on the cell membranes of hDPSCs by cell immunofluorescence. Then, by constructing a lentiviral vector, the expression of Stathmin in hDPSCs was inhibited. Treatment with Stathmin shRNA (shRNA‐Stathmin group) inhibited the ability of hDPSCs to proliferate, as demonstrated by a CCK8 assay and flow cytometry analysis, and suppressed the osteogenic/odontoblastic differentiation ability, as demonstrated by alizarin red S staining and osteogenic/odontoblastic differentiation‐related gene (ALP, BSP, OCN, DSPP) activity, compared to that of hDPSCs from the control shRNA group. Molecular analyses showed that the Shh/GLI1 signalling pathway was inhibited when Stathmin was silenced, and purmorphamine, the Shh signalling pathway activator, was added to hDPSCs in the shRNA‐Stathmin group, real‐time PCR and Western blotting confirmed that expression of Shh and its downstream signalling molecules PTCH1, SMO and GLI1 increased significantly. After activating the Shh signalling pathway, the proliferation of hDPSCs increased markedly, as demonstrated by a CCK8 assay and flow cytometry analysis; osteogenic/odontoblastic differentiation‐related gene (ALP, BSP, OCN, DSPP) expression also increased significantly. Collectively, these findings firstly revealed that Stathmin‐Shh/GLI1 signalling pathway plays a positive role in hDPSC proliferation and osteogenic/odontoblastic differentiation.     

TALE-family homeodomain proteins regulate endodermal sonic hedgehog expression and pattern the anterior endoderm

sonic hedgehog (shh) is expressed in anterior endoderm, where it is required to repress pancreas gene expression and to pattern the endoderm, but the pathway controlling endodermal shh expression is unclear. We find that expression of meis3, a TALE class homeodomain gene, coincides with shh expression in the endoderm of zebrafish embryos. Using a dominant negative construct or anti-sense morpholino oligos (MOs) to disrupt meis3 function, we observe ectopic insulin expression in anterior endoderm. This phenotype is also observed when meis3 MOs are targeted to the endoderm, suggesting that meis3 acts within the endoderm to restrict insulin expression. We also find that meis3 is required for endodermal shh expression, indicating that meis3 acts upstream of shh to restrict insulin expression. Loss of pbx4, a TALE gene encoding a Meis cofactor, produces the same phenotype as loss of meis3, consistent with Meis3 acting in a complex with Pbx4 as reported in other systems. Lastly, we observe a progressive anterior displacement of endoderm-derived organs upon disruption of meis3 or pbx4, apparently as a result of underdevelopment of the pharyngeal region. Our data indicate that meis3 and pbx4 regulate shh expression in anterior endoderm, thereby influencing patterning and growth of the foregut.     

Proteolytic processing yields two secreted forms of sonic hedgehog.

Sonic hedgehog (Shh) is expressed in tissues with known signalling capacities, such as the notochord, the floor plate of the central nervous system, and the zone of polarizing activity in the limb. Several lines of evidence indicate that Shh is involved in floor plate induction, somite patterning, and regulation of anterior-posterior polarity in the vertebrate limb. In this report, we investigate the biochemical behavior of Shh in a variety of expression systems and embryonic tissues. Expression of mouse Shh in Xenopus oocytes, COS cells, and baculovirus-infected insect cells demonstrates that in addition to signal peptide cleavage and N-linked glycosylation, chicken and mouse Shh proteins undergo additional proteolytic processing to yield two peptides with molecular masses of approximately 19 kDa (amino terminus) and 27 kDa (carboxy terminus), both of which are secreted. In transfected COS cells, we show that the 19-kDa peptide does not accumulate significantly in the medium unless heparin or suramin is added, suggesting that this peptide associates with the cell surface or extracellular matrix. This retention appears to depend on sequences in the carboxy-terminal part of the peptide. Finally, detection of the 19-kDa product in a variety of mouse and chicken embryonic tissues demonstrates that the proteolytic processing observed in cell culture is a normal aspect of Shh processing in embryonic development. These results raise the possibility that amino- and carboxyl-terminal regions of Shh may have distinct functions in regulating cell-cell interactions in the vertebrate embryo.     

Spatiotemporal expression of sonic hedgehog signalling molecules in the embryonic mesencephalic dopaminergic neurons

Midbrain dopaminergic neurons (mDA) play an important role in controlling the voluntary motor movement, reward, and emotion-based behaviour. Differentiation of mDA neurons from progenitors depends on several secreted proteins, such as sonic hedgehog (SHH). The present study attempted to elucidate the possible role(s) of some SHH signaling components (Ptch1, Gli1, Gli2 and Gli3) in the spatiotemporal development of mDA neurons along the rostrocaudal axis of the midbrain and their possible roles in differentiation and survival of mDA neurons and the significance of using in vitro models for studying the development of mDA neurons. At E12 and E14, only Ptch1 and Gli1 were expressed in ventrolateral midbrain domains. All examined SHH signalling molecules were not detected in mDA area. Whereas, in MN9D cells, many SHH signalling molecules were expressed and co-localized with the dopaminergic marker; tyrosine hydroxylase (TH), and their expression were upregulated with SHH treatment of the MN9D cells. These results suggest that mDA neurons differentiation and survival might be independent of SHH in the late developmental stages (E12-18). Besides, MN9D cell line is not the ideal in vitro model for investigating the differentiation of mDA and hence, the ventral midbrain primary culture might be favored over MN9D line.     

The role of sonic hedgehog in normal and abnormal craniofacial morphogenesis.

There is growing evidence that implicates a role for Sonic hedgehog (SHH) in morphogenesis of the craniofacial complex. Mutations in human and murine SHH cause midline patterning defects that are manifested in the head as holoprosencephaly and cyclopia. In addition, teratogens such as jervine, which inhibit the response of tissues to SHH, also produce cyclopia. Thus, the loss of SHH signaling during early stages of neural plate patterning has a profound influence of craniofacial morphogenesis. However, the severity of these defects precludes analyses of SHH function during later stages of craniofacial development. We have used an embryonic chick system to study the role of SHH during these later stages of craniofacial development. Using a combination of surgical and molecular experiments, we show here that SHH is essential for morphogenesis of the frontonasal and maxillary processes (FNP and MXPs), which give rise to the mid- and upper face. Transient loss of SHH signaling in the embryonic face inhibits growth of the primordia and results in defects analogous to hypotelorism and cleft lip/palate, characteristics of the mild forms of holoprosencephaly. In contrast, excess SHH leads to a mediolateral widening of the FNP and a widening between the eyes, a condition known as hypertelorism. In severe cases, this widening is accompanied by facial duplications. Collectively, these experiments demonstrate that SHH has multiple and profound effects on the entire spectrum of craniofacial development, and perturbations in SHH signaling are likely to underlie a number of human craniofacial anomalies.     

Perichondrial expression of Wdr5 regulates chondrocyte proliferation and differentiation

Wdr5 is developmentally expressed in osteoblasts and is required for osteoblast differentiation. Mice overexpressing Wdr5 under the control of the mouse α(1)I collagen promoter (Col I-Wdr5) display accelerated osteoblast differentiation as well as accelerated chondrocyte differentiation, suggesting that overexpression of Wdr5 in osteoblasts affects chondrocyte differentiation. To elucidate the molecular mechanism by which overexpression of Wdr5 in the perichondrium regulates chondrocyte differentiation, studies were undertaken using skeletal elements and cultured metatarsals isolated from wild-type and Col I-Wdr5 embryos. FGF18 mRNA levels were decreased in Col I-Wdr5 humeri. Furthermore, local delivery of FGF18 to the bone collar of ex vivo cultures of metatarsals attenuated the chondrocyte phenotype of the Col I-Wdr5 metatarsals. Impairing local FGF action in wild-type metatarsals resulted in a chondrocyte phenotype analogous to that of Col I-Wdr5 metatarsals implicating impaired FGF action as the cause of the phenotype observed. The expression of Twist-1, which regulates chondrocyte differentiation, was increased in Col I-Wdr5 humeri. Chromatin immunoprecipitation analyses demonstrated that Wdr5 is recruited to the Twist-1 promoter. These findings support a model in which overexpression of Wdr5 in the perichondrium promotes chondrocyte differentiation by modulating the expression of Twist-1 and FGF18.     

Forced expression of RNF36 induces cell apoptosis

RNF36 (ring finger protein 36; alias Trif), a member of the RING zinc finger protein family, is expressed in germ cells at round spermatid stages during spermatogenesis. RING finger proteins have been implicated in a variety of functions including oncogenesis, viral replication, and developmental processes. Since no germ cell line is presently available to study the function of RNF36, in this research, we expressed RNF36 truncated and full-length proteins in COS-7 and HEK-293 cell lines to study the effect of RNF36 in somatic cells. The full-length RNF36 protein in both cell lines showed a speckled pattern in the nucleus. Truncated RNF36-1 protein with its putative nuclear localization signal (NLS) remained within the nucleus but lost the speckled pattern. The promyelocytic leukemia (PML) protein, another RING finger protein, was previously identified as present in the nucleus with a speckled pattern. Double-staining and coimmunoprecipitation analyses suggested that RNF36 colocalizes and interacts with PML. In vitro phosphorylation analysis further suggested that RNF36 nuclear localization is under the control of phosphorylation, which might be mediated by p38. Treatment with the p38 inhibitor SB203580 resulted in the cytoplasmic translocation of RNF36. Overexpression of full-length RNF36 in cells induced about half of the transfected cells to undergo cell death. The results of DNA fragmentation assays, flow cytometry assay, and TUNEL staining suggest that the death of RNF36-transfected cells was caused by apoptosis. Following further characterization of the molecular mechanism of RNF36-induced apoptosis, we found that the expression of Bax, caspase-2, and receptor-interacting protein were elevated upon RNF36 induction in test cells. These results suggest that RNF36 may interact with PML and induce cell apoptosis. We suspect that RNF36 may play a role in germ cell homeostasis during spermatogenesis.     

Immunolocalization of sonic hedgehog (Shh) in developing mouse lung.

Expression of sonic hedgehog (Shh) is required for normal development of the lung during embryogenesis. Loss of Shh expression in mice results in tracheoesophageal fistula, lung hypoplasia, and abnormal lung lobulation. To determine whether Shh may play a role later in lung morphogenesis, immunostaining for Shh was performed in mouse lung from embryonic day (E) 10.5 to postnatal day (PD) 24. Shh was detected in the distal epithelium of the developing mouse lung from E10.5 to E16.5. From E16.5 until PD15, Shh was present in epithelial cells in both the peripheral and conducting airways. Although all cells of the developing epithelium uniformly expressed Shh at E10.5, Shh expression was restricted to subsets of epithelial cells by E16.5. Between E16.5 and PD15, non-uniform Shh staining of epithelial cells was observed in the conducting airways in a pattern consistent with the distribution of non-ciliated bronchiolar cells (i.e., Clara cells) and the Clara cell marker CCSP. Shh did not co-localize with hepatocyte nuclear factor/forkhead homologue-4 (HFH-4), beta-tubulin, or with the presence of cilia. These results support the concept that Shh plays a distinct regulatory role in the lung later in morphogenesis, when it may influence formation or cytodifferentiation of the conducting airways.     

The adventures of sonic hedgehog in development and repair. II. Sonic hedgehog and liver development, inflammation, and cancer

Hedgehog (Hh) signaling modulates tissue remodeling by controlling the fate of Hh-responsive cells. Healthy adult livers exhibit little Hh activity. However, cells involved in adult liver repair, including myofibroblasts and progenitors, are capable of producing and responding to Hh ligands. During adult liver injury, Hh ligand production increases and populations of Hh-responsive cells expand. This process is accompanied by fibrosis. Ligand production and Hh-responsive cells diminish as fibrosis resolves and normal hepatic architecture is restored, but Hh signaling persists in hepatocellular carcinomas. These findings suggest that the Hh pathway mediates remodeling responses that are triggered by adult liver damage.     

Prx1 and Prx2 are upstream regulators of sonic hedgehog and control cell proliferation during mandibular arch morphogenesis

The aristaless-related homeobox genes Prx1 and Prx2 are required for correct skeletogenesis in many structures. Mice that lack both Prx1 and Prx2 functions display reduction or absence of skeletal elements in the skull, face, limbs and vertebral column. A striking phenotype is found in the lower jaw, which shows loss of midline structures, and the presence of a single, medially located incisor. We investigated development of the mandibular arch of Prx1(-/-)Prx2(-/-) mutants to obtain insight into the molecular basis of the lower jaw abnormalities. We observed in mutant embryos a local decrease in proliferation of mandibular arch mesenchyme in a medial area. Interestingly, in the oral epithelium adjacent to this mesenchyme, sonic hedgehog (Shh) expression was strongly reduced, indicative of a function for Prx genes in indirect regulation of SHH: Wild-type embryos that were exposed to the hedgehog-pathway inhibitor, jervine, partially phenocopied the lower jaw defects of Prx1(-/-)Prx2(-/-) mutants. In addition, this treatment led to loss of the mandibular incisors. We present a model that describes how loss of Shh expression in Prx1(-/-)Prx2(-/-) mutants leads to abnormal morphogenesis of the mandibular arch.