Concentrations Of Shh Research Articles

Baseline Hedgehog Pathway Activation and Increase of Plasma Wnt1 Protein Are Associated with Resistance to Immune Checkpoint Inhibitors in Advanced Non-Small-Cell Lung Cancer.

Simple SummaryWnt and Hedgehog (Hh) pathways are associated with stemness profile and with resistance to anti-tumor therapies. Pre-clinical data have suggested a role of these pathways in resistance to immune checkpoint inhibitors (ICIs). Here we evaluated the expression and levels of Shh and Wnt molecules both in plasma and tumor samples in a prospective cohort of 63 consecutive NSCLC patients treated with ICIs. We found that baseline Hh activation as assessed by nuclear staining of Gli1 was associated with poor outcome and high primary resistance rate. Increase of plasma Wnt1 during treatment was associated with resistance to ICIs. This is the first study to our knowledge to find an association between the Hh and Wnt pathways and resistance to ICIs in advanced NSCLC patients. We believe that these results are important to understand the molecular mechanisms associated with resistance to ICIs, and ultimately to improve future treatment strategies.Hedgehog (Hh) and Wingless-type (Wnt) pathways are associated with resistance to immune checkpoint inhibitors (ICIs) in preclinical studies. This study aimed to assess the association between expression and activation levels of Wnt and Sonic Hedgehog (Shh) pathways and resistance to ICIs in advanced NSCLC patients treated with ICI. Hh and Wnt pathways activation was assessed by immunohistochemistry (Gli1 and beta-catenin) on corresponding tumor tissues, and by plasma concentrations of Shh and Wnt (Wnt1, Wnt2 and Wnt3) at ICI introduction and at the first clinical evaluation. Sixty-three patients were included, with 36 patients (57.1%) with available tissue. Response rate was lower in Gli1+ NSCLC (20.0%) compared to Gli1 negative (Gli-) NSCLC (55.6%) (p = 0.015). Rate of primary resistance was 69.8%, vs. 31.2%, respectively (p = 0.04), and median progression-free survival (PFS) was 1.9 months (interquartile range (IQR) 1.2–5.7) vs. 6.1 months (1.6–26.0), respectively (p = 0.08). Median PFS and overall survival were shorter in case of increase of Wnt1 concentration during ICI treatment compared to other patients: 3.9 months vs. 11.2 months (p = 0.008), and 15.3 months vs. not reached (p = 0.003). In conclusion, baseline activation of Hh pathway and increase of Wnt1 concentrations during ICI treatment were associated with poor outcome in NSCLC patients treated with ICIs.

Sonic hedgehog signalling as a potential endobronchial biomarker in COPD

BackgroundThe hedgehog (HH) pathway has been associated with chronic obstructive pulmonary disease (COPD) in genome-wide association studies and recent studies suggest that HH signalling could be altered in COPD. We therefore used minimally invasive endobronchial procedures to assess activation of the HH pathway including the main transcription factor, Gli2, and the ligand, Sonic HH (Shh).MethodsThirty non-COPD patients and 28 COPD patients were included. Bronchial brushings, bronchoalveolar lavage fluid (BALF) and bronchial biopsies were obtained from fiberoptic bronchoscopy. Characterization of cell populations and subcellular localization were evaluated by immunostaining. ELISA and RNAseq analysis were performed to identify Shh proteins in BAL and transcripts on lung tissues from non-COPD and COPD patients with validation in an external and independent cohort.ResultsCompared to non-COPD patients, COPD patients exhibited a larger proportion of basal cells in bronchial brushings (26 ± 11% vs 13 ± 6%; p < 0.0001). Airway basal cells of COPD subjects presented less intense nuclear staining for Gli2 in bronchial brushings and biopsies (p < 0.05). Bronchial BALF from COPD patients contained lower Shh concentrations than non-COPD BALF (12.5 vs 40.9 pg/mL; p = 0.002); SHH transcripts were also reduced in COPD lungs in the validation cohort (p = 0.0001).ConclusionThis study demonstrates the feasibility of assessing HH pathway activation in respiratory samples collected by bronchoscopy and identifies impaired bronchial epithelial HH signalling in COPD.

Serum protein expression profiling and bioinformatics analysis in workers occupationally exposed to chromium (VI)

Cr(VI) is widely-recognized as occupational and environmental contaminant, but the precise underlying mechanisms of Cr(VI) induced carcinogenic toxicity remain to be elucidated. Among kinds of toxic mechanisms, alteration of protein profiling usually elaborate a key mechanism of Cr(VI) induced toxicity and carcinogenesis. Large-scale proteins changes can reflect the onset or progression of carcinogenic toxicity, and potential serum protein biomarkers of Cr(VI) exposure. To gain an insight into the serum proteins expression profiling in chromate workers and find potential novel serum proteins biomarkers of Cr(VI) exposure, 107 male participants from a chromate production plant were recruited into the study. Questionnaire was applied to collect personal information and occupational history. Chromium concentration in blood (CrB) was measured to evaluate the participants' internal exposure. Serum proteins profiling and bioinformatics analysis were performed to explore differentially expressed proteins, proteins-chemical interaction network, critical proteins nodes related to the signaling pathways among 16 controls and 25 exposure workers in the first stage. ELISA tests were applied to verify the critical interested proteins nodes in the remaining 41 exposure workers and 25 controls. The results showed that the CrB levels in the control group were significantly lower than that in the exposure group (P<0.05). 44 significantly differentially expressed serum proteins formed 16 significant signaling pathways and a complex proteins-chemical interaction network, which associated with the immune system and extracellular matrix organization. C reactive protein (CRP), sonic hedgehog protein (SHH) and calcium located at critical nodes in proteins-chemical interaction network. There was a significant negative correlation between serum CRP level and CrB (P<0.05), and a significant positive correlation between SHH concentrations and CrB (P<0.05), which indicated that CRP and SHH might be as the potential novel biomarkers of Cr(VI) exposure. Also, the current study preliminarily paved the way to further functional studies to understand the underlying mechanisms and novel serum biomarkers of Cr(VI) exposure.

Patched Receptors Sense, Interpret, and Establish an Epidermal Hedgehog Signaling Gradient

By using the sensitivity of single-molecule fluorescent in situ hybridization, we have precisely quantified the levels and defined the temporal and spatial distribution of Hedgehog signaling activity during embryonic skin development and discovered that there is a Hedgehog signaling gradient along the proximal-distal axis of developing hair follicles. To explore the contribution of Hedgehog receptors Ptch1 and Ptch2 in establishing the epidermal signaling gradient, we quantitated the level of pathway activity generated in Ptch1- and Ptch1;Ptch2-deficient skin and defined the contribution of each receptor to regulation of the levels of Hedgehog signaling identified in wild-type skin. Moreover, we show that both the cellular phenotype and level of pathway activity featured in Ptch1;Ptch2-deficient cells faithfully recapitulates the Peak level of endogenous Hedgehog signaling detected at the base of developing follicles, where the concentration of endogenous Shh is predicted to be highest. Taken together, these data show that both Ptch1 and Ptch2 play a crucial role in sensing the concentration of Hedgehog ligand and regulating the appropriate dose-dependent response.

Taking Shh signaling up a Notch

Notch signaling promotes accumulation of Sonic hedgehog signaling components in the primary cilium to specify ventral neural cell fates.

Mathematical modelling of digit specification by a sonic hedgehog gradient

The three chick wing digits represent a classical example of a pattern specified by a morphogen gradient. Here we have investigated whether a mathematical model of a Shh gradient can describe the specification of the identities of the three chick wing digits and if it can be applied to limbs with more digits. We have produced a mathematical model for specification of chick wing digit identities by a Shh gradient that can be extended to the four digits of the chick leg with Shh-producing cells forming a digit. This model cannot be extended to specify the five digits of the mouse limb. Our data suggest that the parameters of a classical-type morphogen gradient are sufficient to specify the identities of three different digits. However, to specify more digit identities, this core mechanism has to be coupled to alternative processes, one being that in the chick leg and mouse limb, Shh-producing cells give rise to digits; another that in the mouse limb, the cellular response to the Shh gradient adapts over time so that digit specification does not depend simply on Shh concentration.

The Acid-Secreting Parietal Cell as an Endocrine Source of Sonic Hedgehog During Gastric Repair

Sonic Hedgehog (Shh) has been shown to regulate wound healing in various tissues. Despite its known function in tissue regeneration, the role of Shh secreted from the gastric epithelium during tissue repair in the stomach remains unknown. Here we tested the hypothesis that Shh secreted from the acid-secreting parietal cell is a fundamental circulating factor that drives gastric repair. A mouse model expressing a parietal cell-specific deletion of Shh (PC-ShhKO) was generated using animals bearing loxP sites flanking exon 2 of the Shh gene (Shh(flx/flx)) and mice expressing a Cre transgene under the control of the H(+),K(+)-ATPase β-subunit promoter. Shh(flx/flx), the H(+),K(+)-ATPase β-subunit promoter, and C57BL/6 mice served as controls. Ulcers were induced via acetic acid injury. At 1, 2, 3, 4, 5, and 7 days after the ulcer induction, gastric tissue and blood samples were collected. Parabiosis experiments were used to establish the effect of circulating Shh on ulcer repair. Control mice exhibited an increased expression of Shh in the gastric tissue and plasma that correlated with the repair of injury within 7 days after surgery. PC-ShhKO mice showed a loss of ulcer repair and reduced Shh tissue and plasma concentrations. In a parabiosis experiment whereby a control mouse was paired with a PC-ShhKO littermate and both animals subjected to gastric injury, a significant increase in the circulating Shh was measured in both parabionts. Elevated circulating Shh concentrations correlated with the repair of gastric ulcers in the PC-ShhKO parabionts. Therefore, the acid-secreting parietal cell within the stomach acts as an endocrine source of Shh during repair.

Members of the BMP, Shh, and FGF morphogen families promote chicken statoacoustic ganglion neurite outgrowth and neuron survival in vitro

Mechanosensory hair cells of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Members of several morphogen families are expressed within and surrounding the chick inner ear during stages of SAG axon outgrowth and pathfinding. On the basis of their localized expression patterns, we hypothesized that bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), and sonic hedgehog (Shh) may function as guidance cues for growing axons and/or may function as trophic factors once axons have reached their targets. To test this hypothesis, three-dimensional collagen cultures were used to grow Embryonic Day 4 (E4) chick SAG explants for 24 h in the presence of purified proteins or beads soaked in proteins. The density of neurite outgrowth was quantified to determine effects on neurite outgrowth. Explants displayed enhanced neurite outgrowth when cultured in the presence of purified BMP4, BMP7, a low concentration of Shh, FGF8, FGF10, or FGF19. In contrast, SAG neurons appeared unresponsive to FGF2. Collagen gel cultures were labeled with terminal dUTP nick-end labeling and immunostained with anti-phosphohistone H3 to determine effects on neuron survival and proliferation, respectively. Treatments that increased neurite outgrowth also yielded significantly fewer apoptotic cells, with no effect on cell proliferation. When presented as focal sources, BMP4, Shh, and FGFs -8, -10, and -19 promoted asymmetric outgrowth from the ganglion in the direction of the beads. BMP7-soaked beads did not induce this response. These results suggest that a subset of morphogens enhance both survival and axon outgrowth of otic neurons.

Protein kinase Cα and integrin-linked kinase mediate the negative axon guidance effects of Sonic hedgehog.

In addition to its role as a morphogen, Sonic hedgehog (Shh) has also been shown to function as a guidance factor that directly acts on the growth cones of various types of axons. However, the noncanonical signaling pathways that mediate the guidance effects of Shh protein remain poorly understood. We demonstrate that a novel signaling pathway consisting of protein kinase Cα (PKCα) and integrin-linked kinase (ILK) mediates the negative guidance effects of high concentration of Shh on retinal ganglion cell (RGC) axons. Shh rapidly increased Ca(2+) level and activated PKCα and ILK in the growth cones of RGC axons. By in vitro kinase assay, PKCα was found to directly phosphorylate ILK on threonine-173 and -181. Inhibition of PKCα or expression of a mutant ILK with the PKCα phosphorylation sites mutated (ILK-DM), abolished the Shh-induced macropinocytosis, growth cone collapse and repulsive axon turning. In vivo, expression of a dominant negative PKCα or ILK-DM disrupted RGC axon pathfinding at the optic chiasm but not the projection toward the optic disk, supporting that this signaling pathway plays a specific role in Shh-mediated negative guidance effects.

Gastric Sonic Hedgehog Acts as a Macrophage Chemoattractant During the Immune Response to Helicobacter pylori

Macrophages mediate the epithelial response to Helicobacter pylori and are involved in the development of gastritis. Sonic Hedgehog (Shh) regulates gastric epithelial differentiation and function, but little is known about its immunoregulatory role in the stomach. We investigated whether gastric Shh acts as a macrophage chemoattractant during the innate immune response to H pylori infection. Mice with parietal cell-specific deletion of Shh (PC-Shh(KO)) and control mice were infected with H pylori. Levels of gastric Shh, cytokines, and chemokines were assayed by quantitative reverse-transcriptase polymerase chain reaction or by a Luminex-based multiplex assay 2, 7, or 180 days after infection. Circulating concentrations of Shh were measured by enzyme-linked immunosorbent assay. Bone marrow chimera experiments were performed with mice that have myeloid cell-specific deletion of the Hedgehog signal transduction protein Smoothened (LysMCre/Smo(KO)). Macrophage recruitment was measured in gastric tissue and peripheral blood by fluorescence-activated cell sorting analysis. Control mice infected with H pylori for 6 months developed an inflammatory response characterized by infiltration of CD4(+) T cells and increased levels of interferon gamma and interleukin 1β in the stomach. PC-Shh(KO) mice did not develop gastritis, even after 6 months of infection with H pylori. Control mice had increased concentrations of Shh, accompanied by the recruitment of CD11b(+)F4/80(+)Ly6C(high) macrophages 2 days after infection. Control mice that received bone marrow transplants from control mice had an influx of macrophages to the gastric mucosa in response to H pylori infection; this was not observed in H pylori-infected control mice that received bone marrow transplants from LysMCre/Smo(KO) mice. H pylori induces release of Shh from the stomach; Shh acts as a macrophage chemoattractant during initiation of gastritis.

Akt1 interacts with epidermal growth factor receptors and hedgehog signaling to increase stem/transit amplifying cells in the embryonic mouse cortex

A subset of precursors in the embryonic mouse cortex and in neurospheres expresses a higher level of the serine/threonine kinase Akt1 than neighboring precursors. We reported previously that the functional significance of high Akt1 expression was enhanced Akt1 activity, resulting in an increase in survival, proliferation, and self-renewal of multipotent stem/transit amplifying cells. Akt1 can interact with a number of signaling pathways, but the extrinsic factors that are required for specific effects of elevated Akt1 expression have not been identified. In this study we addressed the contributions of signaling via epidermal growth factor (EGF) and hedgehog (Hh) receptors. In EGF receptor-null precursors or following transient inhibition of EGF receptor tyrosine kinase activity, elevating Akt1 by retroviral transduction could still increase survival and proliferation but could not increase self-renewal. We also found that elevated Akt1 expression induced the expression of EGF receptors (EGFRs) in wild-type precursors. Several extrinsic factors, including Shh, can induce EGFR expression by cortical precursors, and we found that elevating Akt1 allowed them to respond to a subthreshold concentration of Shh to induce EGFRs. In precursors that lack the Hh receptor smoothened, however, elevating Akt1 did not increase EGFR expression or self-renewal, though it could still stimulate proliferation. These findings suggest that a subset of precursors in the embryonic cortex that express an elevated level of Akt1 can respond to lower concentrations of Shh than neighboring precursors, resulting in an increase in their expression of EGFRs. Signaling via EGFRs is required for their self-renewal.

A homeodomain feedback circuit underlies step-function interpretation of a Shh morphogen gradient during ventral neural patterning

The deployment of morphogen gradients is a core strategy to establish cell diversity in developing tissues, but little is known about how small differences in the concentration of extracellular signals are translated into robust patterning output in responding cells. We have examined the activity of homeodomain proteins, which are presumed to operate downstream of graded Shh signaling in neural patterning, and describe a feedback circuit between the Shh pathway and homeodomain transcription factors that establishes non-graded regulation of Shh signaling activity. Nkx2 proteins intrinsically strengthen Shh responses in a feed-forward amplification and are required for ventral floor plate and p3 progenitor fates. Conversely, Pax6 has an opposing function to antagonize Shh signaling, which provides intrinsic resistance to Shh responses and is important to constrain the inductive capacity of the Shh gradient over time. Our data further suggest that patterning of floor plate cells and p3 progenitors is gated by a temporal switch in neuronal potential, rather than by different Shh concentrations. These data establish that dynamic, non-graded changes in responding cells are essential for Shh morphogen interpretation, and provide a rationale to explain mechanistically the phenomenon of cellular memory of morphogen exposure.

Activation of sonic hedgehog signaling pathway in S-type neuroblastoma cell lines

The effects of Sonic hedgehog (Shh) signaling pathway activation on S-type neuroblastoma (NB) cell lines and its role in NB tumorigenesis were investigated. Immunohistochemistry was used to detect the expression of Shh pathway components-Patched1 (PTCH1) and Gli1 in 40 human primary NB samples. Western blotting and RT-PCR were used to examine the protein expression and mRNA levels of PTCH1 and Gli1 in three kinds of S-type NB cell lines (SK-N-AS, SK-N-SH and SHEP1), respectively. Exogenous Shh was administrated to activate Shh signaling pathway while cyclopamine was used as a selective antagonist of Shh pathway. S-type NB cell lines were treated with different concentrations of Shh or/and cyclopamine for different durations. Cell viability was measured by using MTT method. Apoptosis rate and cell cycle were assayed by flow cytometry. The xenograft experiments were used to evaluate the role of Shh pathway in tumor growth in immunodeficient mice. High-level expression of PTCH1 and Gli1 was detected in both NB samples and S-type NB cell lines. Cyclopamine decreased the survival rate of the three cell lines while Shh increased it, and the inhibition effects of cyclopamine could be partially reversed by shh pre-treatment. Cyclopamine induced the cell apoptosis and the cell cycle arrest in G0/G1 phase, while Shh induced the reverse effects and could partially prevent effects of cyclopamine. Cyclopamine could also inhibit the growth of NB in vivo. Our studies revealed that activation of the Shh pathway is important for survival and proliferation of S-type NB cells in vivo and in vitro through affecting cell apoptosis and cell cycle, suggesting a new therapeutic approach to NB.

Dynamic assignment and maintenance of positional identity in the ventral neural tube by the morphogen sonic hedgehog.

Author SummaryIn many developing tissues, the pattern in which cell types are generated depends on secreted factors called morphogens. These signalling molecules are produced in specific locations and at specific concentrations, thereby forming concentration gradients. Different target genes are induced at specific distances from the source of the morphogen, and therefore the spatial pattern of gene expression correlates with this concentration gradient. In this study, we examined how cells respond to a morphogen gradient to produce the appropriate pattern of cellular differentiation. We focused on the morphogen Sonic Hedgehog (Shh), which specifies the pattern of different types of neurons in the ventral regions of the neural tube (the embryo's precursor to the central nervous system). We show that in addition to the concentration of Shh, the duration of Shh signalling also contributes to the patterning of the ventral neural tube. A consequence of this is that the genes defining different cellular identities are expressed in a characteristic temporal progression. In addition, sustained Shh signalling is required for more ventral cell types; otherwise they revert to their previous cellular identity. Together these results indicate that dynamic and sustained signalling by Shh is required for the patterning of the ventral neural tube, challenging conventional models of morphogen action that rely solely on the concentration of signal perceived by cells at specific positions in the morphogen gradient.

Urothelium-derived Sonic hedgehog promotes mesenchymal proliferation and induces bladder smooth muscle differentiation

Induction of smooth muscle differentiation from bladder mesenchyme depends on signals that originate from the urothelium. We hypothesize Sonic hedgehog (Shh) is the urothelial signal that promotes bladder mesenchymal proliferation and induces bladder smooth muscle differentiation. Pregnant FVB mice were euthanized on embryonic day (E) 12.5 and fetal bladders were harvested. Two experimental protocols were utilized: (1) Bladder mesenchyme (BLM) was isolated by incubating intact bladders (IB) in 0.02 M EDTA and then removing the urothelium by microdissection. IB and BLM were cultured in Shh-deficient media or BLM was cultured in Shh-supplemented (480 nM) media for 72 h. (2) IB were cultured for 72 h in media containing different concentrations of Shh (0, 48, and 480 nM). Specimens were sized by serial sectioning. Cell counts were performed after trypsin digestion. Immunohistochemistry was performed to detect smooth muscle-specific protein expression. α-Actin expression was quantified using Western blot. All specimens were viable at 72 h. BLM cultured without Shh survived but did not grow or undergo smooth muscle differentiation. IB cultured without Shh and BLM cultured with Shh grew and expressed smooth muscle proteins at 72 h. IB cultured with Shh were larger and contained more cells than IB cultured without Shh (all p<0.05). Increasing Shh concentration from 48 to 480 nM did not change bladder size, cell counts, or the level of α-actin expression. Prior to culture, IB did not express α-actin. After culture of IB in Shh-deficient media, α-actin was detected throughout the mesenchyme except in the submucosal layer. The IB submucosa was thinner after culture with 48 nM Shh and smooth muscle completely obliterated the submucosa after culture with 480 nM Shh. In fetal mouse bladders, urothelium-derived Shh is necessary for mesenchymal proliferation and smooth muscle differentiation. Shh concentration affects mesenchymal proliferation and patterning of bladder smooth muscle.

Sonic hedgehog is a potent chemoattractant for human monocytes: diabetes mellitus inhibits Sonic hedgehog-induced monocyte chemotaxis

The aim of the present study was to evaluate the expression of hedgehog (Hh) signaling molecules and the chemotactic activity of Sonic hedgehog (Shh) in monocytes from control (CTR) and diabetic patients with or without coronary artery disease (CAD). Previously several studies demonstrated that exogenous administration of Shh can induce angiogenesis and accelerate repair of ischemic myocardium and skeletal muscles. Blood samples were collected from (1) CTR (n = 25); (2) patients with stable CAD without diabetes mellitus (CAD−DM, n = 10); and (3) with stable CAD with DM (CAD+DM, n = 15). Monocytes were isolated by Percoll gradient and subjected to PCR and chemotaxis analysis. Hh signaling molecules were expressed in human monocytes, and Shh-induced monocyte chemotaxis. Shh-stimulated migration of monocytes from CTR measured 172.5 ± 90% and a maximal stimulation was observed at Shh concentration of 1 μg/ml. However, Shh failed to induce migration of monocytes from CAD+DM (94.3 ± 27%, P < 0.001 vs. CTR). The impaired response to Shh was associated with strong transcriptional upregulation of the receptor Ptc, while expression of downstream molecules was not altered. Moreover, Ptc is strongly expressed in macrophages of human aortic atherosclerotic plaque. Thus, Shh is a potent chemoattractant for monocytes and it activates classical signaling pathways related to migration. The Shh signaling was negatively affected by DM which might be involved in the pathogenesis of DM-related complications.

Sonic Hedgehog Guides Axons through a Noncanonical, Src-Family-Kinase-Dependent Signaling Pathway

Sonic hedgehog (Shh) plays essential roles in developmental events such as cell fate specification and axon guidance. Shh induces cell fate specification through canonical Shh signaling, mediated by transcription. However, the mechanism by which Shh guides axons is unknown. To study this, we developed an in vitro assay for axon guidance, in which neurons can be imaged while responding to a defined gradient of a chemical cue. Axons of dissociated commissural neurons placed in a Shh gradient turned rapidly toward increasing concentrations of Shh. Consistent with this rapid response, we showed that attraction by Shh does not require transcription. Instead, Shh stimulates the activity of Src family kinase (SFK) members in a Smoothened-dependent manner. Moreover, SFK activity is required for Shh-mediated guidance of commissural axons, but not for induction of Gli transcriptional reporter activity. Together, these results indicate that Shh acts via a rapidly acting, noncanonical signaling pathway to guide axons.

Sonic hedgehog promotes porcine oocyte maturation and early embryo development

In the present study, we investigated the effects of the Sonic hedgehog (Shh) protein on porcine oocyte maturation and early embryo development. Immunohistochemistry showed activation of Shh signalling in cumulus-oocyte complexes (COCs), as reflected by Patched (Ptc), Smoothened (Smo) and Gli1 expression in oocytes, cumulus cells and granulosa cells, particularly those of small follicles (<2 mm in diameter). Western blot analysis showed Smo expression in COCs and in denuded oocytes derived from small and medium (3-7 mm)-sized follicles. Small follicles contained the highest concentration of Shh in follicular fluid compared with medium-sized and large (>7 mm in diameter) follicles. Supplementation with Shh (0.5 or 1 microg mL(-1)) enhanced oocyte maturation compared with the control group (92.4% and 90.4% v. 81.9%, respectively; P < 0.05). This effect was reversed by the simultaneous addition of cyclopamine (1-2 microm), an Shh inhibitor. Similar to intact COCs, denuded COCs showed enhanced maturation following Shh supplementation. Furthermore, cyclin B1 content, extracellular signal-regulated kinase 1/2 phosphorylation, intracellular calcium release, blastocyst rate and total cell numbers were greater (P < 0.05) in oocytes matured in the presence of 0.5 and 1 microg mL(-1) Shh compared with control oocytes. The findings of the present study provide the first evidence that the Shh signalling pathway is active, or at least partially activated, in the porcine ovary and is likely to promote oocyte cytoplasmic and nuclear maturation, as well as subsequent in vitro development, although the underlying mechanisms remain to be elucidated.

Multivalency of Sonic Hedgehog Conjugated to Linear Polymer Chains Modulates Protein Potency

A potently active multivalent form of the protein Sonic hedgehog (Shh) was produced by bioconjugation of a modified recombinant form of Shh to the linear polymers poly(acrylic acid) (pAAc) and hyaluronic acid (HyA) via a two-step reaction exploiting carboimiide and maleimide chemistry. Efficiency of the conjugation was approximately 75% even at stoichiometric ratios of 30 Shh molecules per linear HyA chain (i.e., 30:1 Shh/HyA). Bioactivity of the conjugates was tested via a cellular assay across a range of stoichiometric ratios of Shh molecules to HyA linear chains, which was varied from 0.6:1 Shh/HyA to 22:1 Shh/HyA. Results indicate that low conjugation ratios decrease Shh bioactivity and high ratios increase this activity beyond the potency of monomeric Shh, with approximately equal activity between monomeric soluble Shh and conjugated Shh at 7:1 Shh/HyA. In addition, high-ratio constructs increased angiogenesis determined by the in vivo chick chorioallantoic membrane (CAM) assay. These results are captured by a kinetic model of multiple interactions between the Shh/HyA conjugates and cell surface receptors resulting in higher cell signaling at lower bulk Shh concentrations.

Region-specific requirement for cholesterol modification of sonic hedgehog in patterning the telencephalon and spinal cord

Sonic hedgehog (Shh) secreted from the axial signaling centers of the notochord and prechordal plate functions as a morphogen in dorsoventral patterning of the neural tube. Active Shh is uniquely cholesterol-modified and the hydrophobic nature of cholesterol suggests that it might regulate Shh spreading in the neural tube. Here, we examined the capacity of Shh lacking the cholesterol moiety (ShhN) to pattern different cell types in the telencephalon and spinal cord. In mice expressing ShhN, we detected low-level ShhN in the prechordal plate and notochord, consistent with the notion that ShhN can rapidly spread from its site of synthesis. Surprisingly, we found that low-level ShhN can elicit the generation of a full spectrum of ventral cell types in the spinal cord, whereas ventral neuronal specification and ganglionic eminence development in the Shh(N/-) telencephalon were severely impaired, suggesting that telencephalic patterning is more sensitive to alterations in local Shh concentration and spreading. In agreement, we observed induction of Shh pathway activity and expression of ventral markers at ectopic sites in the dorsal telencephalon indicative of long-range ShhN activity. Our findings indicate an essential role for the cholesterol moiety in restricting Shh dilution and deregulated spread for patterning the telencephalon. We propose that the differential effect of ShhN in patterning the spinal cord versus telencephalon may be attributed to regional differences in the maintenance of Shh expression in the ventral neuroepithelium and differences in dorsal tissue responsiveness to deregulated Shh spreading behavior.