Sonic hedgehog signaling is associated with resistance to zoledronic acid in CD133high/CD44high prostate cancer stem cells

Molecular Biology Reports - Cancer stem cells (CSCs) are a unique population that has been linked to drug resistance and metastasis and recurrence of prostate cancer. The sonic hedgehog (SHH)...     

Sonic hedgehog signaling is required during the appearance of spinal cord oligodendrocyte precursors

Spinal cord oligodendrocyte precursors arise in the ventral ventricular zone as a result of local signals. Ectopic oligodendrocyte precursors can be induced by sonic hedgehog (Shh) in explants of chick dorsal spinal cord over an extended developmental period. The role of Shh during normal oligodendrocyte development is, however, unclear. Here we demonstrate that Shh is localized to the ventral spinal cord immediately prior to, and during the appearance of oligodendrocyte precursors. Continued expression of Shh is required for the appearance of spinal cord oligodendrocyte precursors as neutralization of Shh signaling both in vivo and in vitro during a defined developmental period blocked their emergence. The inhibition of oligodendrocyte precursor emergence in the absence of Shh signaling was not the result of inhibiting precursor cell proliferation, and the neutralization of Shh signaling after the emergence of oligodendrocyte precursors had no effect on the appearance of additional cells or their subsequent differentiation. Similar concentrations of Shh induce motor neurons and oligodendrocytes in dorsal spinal cord explants. However, in explants from early embryos the motor neuron lineage is preferentially expanded while in explants from older embryos the oligodendrocyte lineage is preferentially expanded.     

Sonic hedgehog signaling is critical for cytodifferentiation and cusp formation in developing mouse molars

The present study was designed to investigate the direct role of Shh molecule on cytodifferentiation and cusp formation. Affi-gel blue beads soaked in exogenous Shh-N, Shh antibody or BSA control protein were implanted between the epithelium and mesenchyme of isolated molar germs at the cap stage. The recombinants were grafted for culture under the kidney capsules respectively. In compared to the control, additional Shh-N protein could not enhance the ameloblasts and odontoblasts differentiation of the explanted tooth germs. While, application of Shh antibody retarded these events. After 4 weeks of subrenal culture, the teeth dissected from the explants treated with Shh-N were multicuspid. Most of the teeth harvested from the Shh antibody group were small and single irregularly shaped cusp was visible. The main cusp height in this group was reduced. The results indicated Shh signaling pathway is critical for odontoblast and ameloblast differentiation and patterns cusp formation. Lu Zhang and Fang Hua contribute equally.     

Infant formula ingestion is associated with the development of diabetes in the BB/Wor rat

The association between early exposure to cow's milk products in infancy and risk for insulin dependent diabetes mellitus (IDDM) is controversial. We examined whether the ingestion of cow's milk-based infant formula altered the expression of the diabetic syndrome in the BB/Wor rat, an animal model of IDDM. Pregnant BB/Wor dams were obtained from the NIH contract colony at the University of Massachusetts and housed under semi-barrier conditions. Rat pups were intubated with 1 to 2 ml of commercially available cow's milk-based infant formula (Enfamil or Nutramigen) or sham intubated (controls) daily from day 12 to day 25 of life. Pups were weaned at day 25 and monitored for glucosuria daily through 120 days of life. All rats including dams consumed a milk-free rat chow and acidified water ad libitum throughout the study. The mean age of disease onset was 4 to 10 days earlier in Nutramigen-fed and Enfamil-fed rats relative to controls (84+/-3, 78+/-2 and 88+/-4 days, respectively); the mean age of disease onset was significantly different between controls and Enfamil-fed animals (p<0.05). At 120 days, 60% (12/20) of control rats developed diabetes versus 100% of animals fed either type of infant formula prior to weaning (15/15:Enfamil-fed; 19/19:Nutramigen-fed) (p<0.05). These data indicate that direct, early ingestion of cow's milk-based formula was related to the expression of diabetes in the BB/Wor rat.     

Genetic analysis of the BB/W diabetic rat

A large colony of BB/W diabetic rats has been developed as a research model for insulin dependent, type 1 diabetes mellitus. The foundation stock had 8% diabetics which appeared in a sporadic manner. The Worcester (W) colony was inbred by brother X sister matings for 11 generations and the proportion of diabetics increased to over 50%. The age of detection varies from 46 to 250 days. For selection purposes, classification was made at 120 days, which means that 15 to 20% potential diabetics were classified as normal. Evidence from different analyses indicates that the inheritance of diabetes is by a recessive gene or gene cluster with 50% penetrance at 120 days. The selection of breeding stock from diabetic parents raised the proportion of diabetics produced by two normal parents from 12 to 43%. Diallel tests show that diabetic and normal offspring of two diabetic parents have the same diabetic genotype. Outcrosses to other strains of rat indicate that the trait is transferred as a recessive with only 3% diabetics recovered in the F2 where noninbred BB stock was used as the diabetic source, and 36% where partially inbred BB/W was used as the diabetic parent. Since the proportion of diabetics produced by all types of crosses has changed, and may continue to change with changes in the genetic background, we have used the operational term penetrance to describe the frequency of diabetes in individuals homozygous for the diabetes gene cluster. At present the penetrance at 120 days is 59% in the BB/W colony.     

A neural crest deficit in Down syndrome mice is associated with deficient mitotic response to Sonic hedgehog

Trisomy 21 results in phenotypes collectively referred to as Down syndrome (DS) including characteristic facial dysmorphology. Ts65Dn mice are trisomic for orthologs of about half of the genes found on human chromosome 21 and exhibit DS-like craniofacial abnormalities, including a small dysmorphic mandible. Quantitative analysis of neural crest (NC) progenitors of the mandible revealed a paucity of NC and a smaller first pharyngeal arch (PA1) in Ts65Dn as compared to euploid embryos. Similar effects in PA2 suggest that trisomy causes a neurocristopathy in Ts65Dn mice (and by extension, DS). Further analyses demonstrated deficits in delamination, migration, and mitosis of trisomic NC. Addition of Sonic hedgehog (Shh) growth factor to trisomic cells from PA1 increased cell number to the same level as untreated control cells. Combined with previous demonstrations of a deficit in mitogenic response to Shh by trisomic cerebellar granule cell precursors, these results implicate common cellular and molecular bases of multiple DS phenotypes.     

Endothelins urinary excretion is increased in spontaneously diabetic rats: BB/BB

Previously we reported (1) an increase of endothelin-1,2 (ET) content, in urine of rats made diabetic with streptozotocin (STZ), starting three days and up to 20 weeks from diabetes induction. The increased ET excretion was considered as an early marker of endothelial damage. To ascertain if this phenomenon was present also in a strain of spontaneously diabetic rats, endothelin-1,2 urinary excretion was determined in BB/BB diabetic rats, and their control (BB/WB), at different times after the onset of diabetes, (two, four, six and twelve weeks). BB/BB diabetic rats showed elevated urinary excretion of endothelins as compared to BB/WB control rats, starting two weeks after diabetes onset, and up to twelve weeks. In the same animals, Nerve Conduction Velocity (NCV), was monitored at the same time as an index of the occurrence of a diabetes complication (peripheral neuropathy). NCV resulted to be impaired in the BB/BB diabetic rats as compared to control rats; however the increase of ET in urine, is earlier in comparison to peripheral neuropathy. These data suggest the hypothesis that endothelial damages preceed the overt manifestations of peripheral neuropathy associated to diabetes.     

Sonic hedgehog is involved in osteoblast differentiation by cooperating with BMP-2

The roles of Sonic hedgehog (Shh) and Bone morphogenetic protein-2 (Bmp-2) in osteoblast differentiation were investigated using in vitro cell systems. Recombinant amino-terminal portion of SHH (rSHH-N) dose dependently stimulated ALP activity in C3H10T1/2 and MC3T3-E1 cells. rSHH-N induced expression of Osteocalcin mRNA in C3H10T1/2 cells. A soluble form of the receptor for type IA BMP receptor antagonized rSHH-N-induced ALP activity in C3H10T1/2 and MC3T3-E1 cells, indicating that BMPs are involved in SHH-induced osteoblast differentiation. Simultaneous supplement with rSHH-N and BMP-2 synergistically induced ALP activity and expression of Osteocalcin mRNA in C3H10T1/2 cells. Pretreatment with rSHH-N for 6 h enhanced the response to BMP-2 by increasing ALP activity in C3H10T1/2 and MC3T3-E1 cells. Stimulatory effects of rSHH-N and additive effects with rSHH-N and BMP-2 on ALP activity were also observed in mouse primary osteoblastic cells. Transplantation of BMP-2 (1 microg) into muscle of mice induced formation of ectopic bone, whereas transplantation of r-SHH-N (1-5 microg) failed to generate it. These results indicate that Shh plays important roles in osteoblast differentiation by cooperating with BMP.     

Temporal and spatial effects of Sonic hedgehog signaling in chick eye morphogenesis

Proper dorsal--ventral pattern formation of the optic cup is essential for vertebrate eye morphogenesis and retinotectal topographic mapping. Previous studies have suggested that midline tissue-derived Sonic hedgehog (Shh) molecules play critical roles in establishing the bilateral eye fields and in determining the proximal--distal axis of the eye primordium. Here, we have examined the temporal requirements for Shh during the optic vesicle to optic cup transition and after early optic cup formation in chick embryos. Both misexpressing Shh by virus and blocking Shh activity by antibodies resulted in disruption of ventral ocular tissues. Decreasing endogenous Shh signals unexpectedly revealed a sharp morphological boundary subdividing dorsal and ventral portions of the optic cup. In addition, Shh signals differentially influenced expression patterns of genes involved in ocular tissue specification (Pax6, Pax2, and Otx2) and dorsal--ventral patterning (cVax) within the ventral but not dorsal optic cup. Ectopic Shh suppressed expression of Bone Morphogenetic Protein 4 (BMP4) in the dorsal retina, whereas reducing endogenous Sonic hedgehog activity resulted in a ventral expansion of BMP4 territory. These results demonstrate that temporal requirements for Shh signals persist after the formation of the optic cup and suggest that the early vertebrate optic primordium may be subdivided into dorsal and ventral compartments. We propose a model in which ventrally derived Shh signals and dorsally restricted BMP4 signals act antagonistically to regulate the growth and specification of the optic primordium.     

Altered SR protein expression associated with contractile dysfunction in diabetic rat hearts

The goal of this study was to examine whether alteration of sarcoplasmic reticulum (SR) protein levels is associated with early-onset diastolic and late-onset systolic dysfunction in streptozotocin (STZ)-induced diabetic rat hearts. Four-week diabetic rat hearts exhibited slow relaxation, whereas 6-wk diabetic rat hearts exhibited slow and depressed contraction. Total phospholamban level was increased, and phosphorylated level was decreased in 4- and 6-wk diabetic rat hearts. Sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2) protein level was unchanged in 4-wk but decreased in 6-wk diabetic rat hearts. Only the apparent affinity of SR Ca2+ uptake for Ca2+ was decreased in 4-wk diabetic rat hearts, but the apparent affinity and the maximum rate was decreased in 6-wk diabetic rat hearts. Insulin treatment of the diabetic rats normalized SR protein expression and function. It was concluded that an increase in nonphosphorylated phospholamban and a decrease in the apparent affinity of SR Ca2+ pump for Ca2+ are associated with early-onset diastolic dysfunction and decreases in SERCA2 protein level and apparent affinity and maximum velocity of SR Ca2+ pump are associated with late-onset systolic dysfunction in diabetic rats.     

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.     

Sonic hedgehog is associated with H+-K+-ATPase-containing membranes in gastric parietal cells and secreted with histamine stimulation

Sonic hedgehog (Shh) is found within gastric parietal cells and processed from a 45-kDa to a 19-kDa bioactive protein by an acid- and protease-dependent mechanism. To investigate whether Shh is associated with the parietal cell membrane compartment that becomes exposed to both acid and proteolytic enzymes during acid secretion, the cellular location of Shh within resting and stimulated gastric parietal cells was examined. Immunofluorescence microscopy of rabbit stomach sections showed that Shh colocalized predominantly with parietal and pit, not chief/zymogen or neck, cell markers. In resting and histamine-stimulated rabbit gastric glands Shh was expressed only in parietal cells close to H+-K+-ATPase-containing tubulovesicular and secretory membranes with some colocalizing with gamma-actin at the basolateral membrane. Gastric gland microsomal membranes were prepared by differential and sucrose gradient centrifugation and immunoisolation with an anti-H+-K+-ATPase-alpha subunit antibody. The 45- and 19-kDa Shh proteins were detected by immunoblot in immunopurified H+-K+-ATPase-containing membranes from resting and stimulated gastric glands, respectively. Incubating glands with a high KCl concentration removed Shh from the membranes. Histamine stimulated 19-kDa Shh secretion from gastric glands into the medium. In human gastric cancer 23132/87 cells cultured on permeable membranes, histamine increased 19-kDa Shh secretion into both apical and basolateral media. These findings show that Shh is a peripheral protein associated with resting and stimulated H+-K+-ATPase-expressing membranes. In addition, Shh appears to be expressed at or close to the basolateral membrane of parietal cells.     

Sonic hedgehog signaling in forebrain development and its interactions with pathways that modify its effects

During the development of the nervous system and other organs in the embryo, a limited set of master signaling pathways are used repeatedly for induction, patterning and growth. Among these, the Sonic hedgehog (Shh) pathway is crucial for the development of many structures in the brain. How the context-specific interplay between these various signaling pathways produces distinct temporal and spatial outcomes is not clear. Resolving this problem is a major goal in the study of cell and organ development. Here, we focus on signaling events during dorso-ventral patterning of the embryonic forebrain in vertebrates. In particular, we discuss the role of the Shh pathway in this process and on its interactions with the FGF, retinoic acid and Nodal pathways and other information cascades that modify its effects.     

Sonic hedgehog is required for vascular outgrowth in the hindbrain choroid plexus

Critical to the exchange and metabolic functions served by tissues like brain choroid plexi and lung is the coherent development of an epithelial sheet of large surface area in tight apposition to an extensive vascular bed. Here, we present functional experiments in the mouse demonstrating that Sonic hedgehog (Shh) produced by hindbrain choroid plexus epithelium induces the extensive vascular outgrowths and vascular surface area fundamental to choroid plexus functions, but does not induce the more specialized endothelial cell features of fenestrations and bore size. Our findings indicate that these Shh-dependent vascular elaborations occur even in the presence of Vegf and other established angiogenic factors, suggesting either that the levels of these factors are inadequate in the absence of Shh or that a different set of factors may be more essential to choroid plexus outgrowth. Transducing the Shh signal is a perivascular cell—the pericyte—rather than the more integral vascular endothelial cell itself. Moreover, our findings suggest that hindbrain choroid plexus endothelial cells, as compared to other vascular endothelial cells, are more dependent upon pericytes for instruction. Thus, in addition to Shh acting on the progenitor pool for choroid plexus epithelial cells, as previously shown, it also acts on choroid plexus pericytes, and together serves the important role of coordinating the development of two disparate yet functionally dependent structures—the choroid plexus vasculature and its ensheathing epithelium.     

The regulation of tooth morphogenesis is associated with epithelial cell proliferation and the expression of Sonic hedgehog through epithelial-mesenchymal interactions

Ectodermal organs, such as the tooth, salivary gland, hair, and mammary gland, develop through reciprocal epithelial–mesenchymal interactions. Tooth morphologies are defined by the crown width and tooth length (macro-morphologies), and by the number and locations of the cusp and roots (micro-morphologies). In our current study, we report that the crown width of a bioengineered molar tooth, which was reconstructed using dissociated epithelial and mesenchymal cells via an organ germ method, can be regulated by the contact area between epithelial and mesenchymal cell layers. We further show that this is associated with cell proliferation and Sonic hedgehog (Shh) expression in the inner enamel epithelium after the germ stage has formed a secondary enamel knot. We also demonstrate that the cusp number is significantly correlated with the crown width of the bioengineered tooth. These findings suggest that the tooth micro-morphology, i.e. the cusp formation, is regulated after the tooth width, or macro-morphology, is determined. These findings also suggest that the spatiotemporal patterning of cell proliferation and the Shh expression areas in the epithelium regulate the crown width and cusp formation of the developing tooth.     

Sonic hedgehog signaling at gastrula stages specifies ventral telencephalic cells in the chick embryo

A secreted signaling factor, Sonic hedgehog (Shh), has a crucial role in the generation of ventral cell types along the entire rostrocaudal axis of the neural tube. At caudal levels of the neuraxis, Shh is secreted by the notochord and floor plate during the period that ventral cell fates are specified. At anterior prosencephalic levels that give rise to the telencephalon, however, neither the prechordal mesoderm nor the ventral neural tube expresses Shh at the time that the overt ventral character of the telencephalon becomes evident. Thus, the precise role and timing of Shh signaling relevant to the specification of ventral telencephalic identity remains unclear. By analysing neural cell differentiation in chick neural plate explants we provide evidence that neural cells acquire molecular properties characteristic of the ventral telencephalon in response to Shh signals derived from the anterior primitive streak/Hensen's node region at gastrula stages. Exposure of prospective anterior prosencephalic cells to Shh at this early stage is sufficient to initiate a temporal program of differentiation that parallels that of neurons generated normally in the medial ganglionic eminence subdivision of the ventral telencephalon.     

Progenitor cell proliferation in the retina is dependent on Notch-independent Sonic hedgehog/Hes1 activity

Sonic hedgehog (Shh) is an indispensable, extrinsic cue that regulates progenitor and stem cell behavior in the developing and adult mammalian central nervous system. Here, we investigate the link between the Shh signaling pathway and Hes1, a classical Notch target. We show that Shh-driven stabilization of Hes1 is independent of Notch signaling and requires the Shh effector Gli2. We identify Gli2 as a primary mediator of this response by showing that Gli2 is required for Hh (Hedgehog)-dependent up-regulation of Hes1. We also show using chromatin immunoprecipitation that Gli2 binds to the Hes1 promoter, which suggests that Hes1 is a Hh-dependent direct target of Gli2 signaling. Finally, we show that Shh stimulation of progenitor proliferation and cell diversification requires Gli2 and Hes1 activity. This paper is the first demonstration of the mechanistic and functional link between Shh, Gli, and Hes1 in the regulation of progenitor cell behavior.     

Structural basis for catalyzed assembly of the Sonic hedgehog–Patched1 signaling complex

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