Shh Secretion Research Articles

Role of Microparticles as Messengers Enhancing Stem Cell Activity After Genetic Engineering

Despite significant therapeutic advances over several decades, myocardial infarction remains the most common cause of death worldwide. In this context, cell-based therapy was considered a promising novel strategy for regeneration of healthy, functionally integrated, myocardial tissue. Unfortunately, the clinical trials using cell-based therapy did not always confirm the preclinical experimental results.1,2 Therefore, in the last decade, the efforts were focused on studying the complex molecular and cellular mechanisms contributing to improved myocardial remodeling and function after stem cell transplantation.3 Article, see p 312 Because cell-based therapy has been proposed, various cell types were tested in transplantation experiments.4–10 Among all, autologous bone marrow-derived CD34+ stem cells demonstrate a major benefit, showing favorable impact on angiogenic growth, without the toxicity and inflammatory response observed in nonautologous cell-based therapy.11,12 However, insufficient number of donor stem cells, low cell viability, and inefficient expansion techniques limit the effects of this therapy. In this regard, overexpressing angiogenic growth factors in transplanted cells has been reported to be beneficial by attenuating myocardial ischemia and preserving the heart function.13–17 In this issue of Circulation Research , Mackie et al investigate the mechanisms of preserving the heart function after transplantation of CD34+ cells overexpressing sonic hedgehog (Shh) protein, a well-established angiogenic factor.18 After …

Abstract 2567: DIM regulates anoikis through sonic hedgehog in ovarian cancer cells

Abstract Ovarian cancer is the second leading gynecological cancer with poor prognosis. By the time of detection, it usually metastasizes to peritoneum or organs such as stomach and liver. Detachment of a cell from extracellular matrix (ECM) allows the cells to travel and lodge at metastatic site. Anoikis is an emerging form of cell death that is associated with the apoptosis of cells detached from ECM. Normal or immortal epithelial cells are sensitive to anoikis. However, carcinomas are generally associated with resistance to anoikis. Hence, induction of anoikis is an important step in preventing metastasis. We have recently shown that diindolylmethane (DIM), a compound present in cruciferous vegetables reduces the survival of ovarian cancer cells by inducing apoptosis. Here, we evaluated the effect of DIM in inducing anoikis in ovarian cancer cells. A2780, SKOV-3 and OVCAR-429 cells were grown on low attachment plates and treated with various concentrations of DIM for 24 hours and analyzed. Our results show that DIM treatment resulted in the concentration-dependent anoikis in all the cell lines. For example, anoikis induction was approximately 50-70% in all the cell lines treated with 75µM DIM. Furthermore, Integrin Linked Kinase 1 (ILK-1), an important regulator of anoikis was significantly down-regulated by DIM treatment. Western blotting revealed cleavage of caspase-3 and PARP by DIM treatment, confirming anoikis induction in our model. Few recent studies showed that Sonic Hedgehog (Shh) pathway is activated in ovarian tumors. Shh pathway is also associated with metastasis of various cancers. We therefore next determined whether Shh pathway was involved in anoikis induction by DIM. Our results demonstrated that DIM treatment reduced the expression of Shh and Gli1 in a concentration-dependent manner. Moreover, Shh secretion as well was reduced by DIM treatment in ovarian cancer cells. Importantly, Shh pretreatment significantly inhibited DIM-induced anoikis in A2780 ovarian cancer cells. Treatment of cells with Shh also reduced the cleavage of caspase-3 or PARP induced by DIM treatment indicating the involvement of Shh pathway in anoikis. Taken together, these results suggest that DIM induces anoikis in ovarian cancer cells by regulating sonic hedgehog signaling. Further in-depth mechanistic studies are in progress to establish anoikis induction by DIM and to validate it in an in vivo model. [Supported in part by R01 grants CA 1065953 and CA 129038 awarded to S.K.S by National Cancer Institute, NIH] Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2567. doi:1538-7445.AM2012-2567

UP‐REGULATION OF TFAP2C BY FGF DURING SHH INDUCTION IN LIMB BUDS

During development, limb outgrowth has been linked to a reciprocal feedback loop between Fibroblast growth factors (Fgfs) and Sonic Hedgehog (Shh). Fgfs from the apical ectodermal ridge (AER) at the distal tip of the limb bud promote secretion of Shh from the zone of polarizing activity (ZPA), a cluster of cells in the distal posterior aspect of limb bud mesoderm. In turn, Shh induces Fgf from the posterior AER. This reciprocal loop is also critical to limb regeneration, and previous studies have shown that Shh up‐regulation correlates with the capacity of Fgfs to accomplish regenerative limb outgrowth. Using gene arrays, we have identified a number of potential Fgf downstream targets in the Fgf‐Shh pathway. In this report, we examined the expression of Transcription factor AP‐2 gamma (Tfap2c).To determine the time course of Fgf‐induced Tfap2c up‐regulation, we implanted Fgf‐soaked beads into stage 23 chick wing buds and evaluated Tfap2c expression by in situ hybridization at several time points.Tfap2c was up‐regulated within 3 hours of Fgf exposure; however, treatment with the protein synthesis inhibitor cycloheximide abolished induction.We conclude that Tfap2c is a close, but indirect, downstream target of Fgf during limb development and regeneration. Further studies are needed to determine whether Tfap2c participates in the Fgf‐Shh pathway or plays an alternative role in limb outgrowth.Grant Funding Source: departmental

Sonic hedgehog maintains survival and growth of chronic myeloid leukemia progenitor cells through β-catenin signaling

Sonic hedgehog (Shh) signaling plays an important role in many human cancers and cancer stem cells. Here we investigate the activity and functional role of Shh signaling in chronic myeloid leukemia (CML) and leukemia progenitor cells. Differential activation of Shh signaling was found in about 50% CML chronic phase samples, about 70% of CML accelerated phase samples, and >80% CML blast crisis phase samples. Deregulated activation of Shh signaling was observed in CD34(+) and c-kit(+) leukemia progenitor cells. Stimulation of Shh signaling with exogenous Shh peptide induced expansion of CD34(+) and c-kit(+) progenitor cells (p < 0.05), inversely, blocking the pathway with signal inhibitor induced cell apoptosis (p < 0.05). Low level of Shh protein was observed in CML bone marrow stromal cells, and CD34(+) progenitor cells are less sensitive to exogenous Shh peptide and more sensitive to cyclopamine than CD34(-) cells (p < 0.05), implying cell-autonomous activation of Shh signaling play a predominant role in progenitor cells. Coactivation of Shh and β-catenin signaling was found in CD34(+) and c-kit(+) progenitor cells. Administration of Shh-neutralizing antibody or Wnt3a-neutralizing antibody in c-kit(+) progenitor cells induced cell apoptosis; however, Wnt3a peptide could salvage cell apoptosis, while Shh peptide failed to revert anti-Wnt3a-induced cell apoptosis. C-MYC, GLI1, BCL-2, and P21 were also found to be downstream targets of Shh signaling, mediating apoptosis or G(2)/M cell cycle arrest of progenitor cells. Our results demonstrate that autoactivated Shh signaling provides survival and proliferative cues in CML progenitor cells through downstream β-catenin signaling, suggesting a novel therapeutic approach in CML.

Processing-dependent trafficking of Sonic hedgehog to the regulated secretory pathway in neurons

Neurons are an important source of the secreted morphogen Sonic hedgehog (Shh), however, little is known about neuron-specific regulation of Shh transport and secretion. To study this process, we investigated the subcellular distribution of Shh in primary neurons and differentiated cells of a neuroendocrine cell line by fluorescence microscopy and biochemical fractionation. In retinal ganglion cells, endogenous Shh was distributed as intra- and extracellular puncta at the soma, dendrites, axons and neurite terminals. Shh + puncta move bidirectionally and colocalize with markers of synaptic vesicles (SVs) and dense core granules. Lipid modification and proteolysis were required for Shh sorting to SVs and cell surface association. Finally, consistent with its association with regulated secretory vesicles, Shh secretion could be induced under depolarizing conditions. Taken together, these observations suggest that long-range Shh transport and signalling in neurons involves trafficking to the regulated secretory pathway and cell surface accumulation of Shh on axons and suggests a link between neuronal activity and Shh release.

Sonic Hedgehog signaling mediates bone marrow‐derived mesenchymal stem cell proliferation and recruitment

ObjectiveBone marrow‐derived mesenchymal stem cells (MSCs) are recruited to the site of chronic inflammation during gastric cancer progression. Given that increased interferon‐gamma (IFNγ) and Sonic Hedgehog (Shh) expression contribute to cancer development, the role of the Shh signaling pathway as a mediator of IFNγ induced proliferation and recruitment of MSCs was studied.MethodsMSCs transduced with lentiviral particles expressing shRNA for Shh (MSCsShhKO) or empty vector (MSCsVect) were analyzed for cell cycle changes by flow cytometry. In vivo, MSCs tagged with RFP were transferred (via tail vein injection) into C57BL/6 mice and treated with either PBS or IFNγ (1μg/mouse/day) for 14 days. To identify the mechanism of recruitment, chemokine SDF‐1α was used in a chemotaxis assay with MSCsShhKO and MSCsVect.ResultsIFNγ significantly increased MSC proliferation, a response mediated by the secretion of Shh from cultured cells and blocked by Hedgehog signaling inhibitor cyclopamine. MSCsShhKO were arrested in G0/G1 phase compared to MSCsVect. Mice injected with IFNγ showed significant recruitment of RFP‐tagged MSCs to the gastric mucosa. Compared to MSCsVect, MSCsShhKO failed to migrate toward the SDF‐1α reflected by decreased CXCR4 expression.ConclusionIFNγ induces proliferation and initiates recruitment of MSCs, a mechanism mediated by Shh signaling.(Supported by start‐up funds, Zavros)

Evidence for a role of vertebrate Disp1 in long-range Shh signaling

Dispatched 1 (Disp1) encodes a twelve transmembrane domain protein that is required for long-range sonic hedgehog (Shh) signaling. Inhibition of Disp1 function, both by RNAi or dominant-negative constructs, prevents secretion and results in the accumulation of Shh in source cells. Measuring the Shh response in neuralized embryoid bodies (EBs) derived from embryonic stem (ES) cells, with or without Disp1 function, demonstrates an additional role for Disp1 in cells transporting Shh. Co-cultures with Shh-expressing cells revealed a significant reduction in the range of the contact-dependent Shh response in Disp1(-/-) neuralized EBs. These observations support a dual role for Disp1, not only in the secretion of Shh from the source cells, but also in the subsequent transport of Shh through tissue.

Sonic hedgehog ligand partners with caveolin-1 for intracellular transport

Prenatal alcohol exposure is the most common environmental factor leading to congenital birth defects in the United States. Although significant progress has been made in this field, the detailed molecular pathology of fetal alcohol syndrome (FAS) remains to be determined. Previously, we have shown that alcohol exposure perturbs hedgehog signal transduction in zebrafish embryos by inhibiting the post-translational cholesterol modification of Sonic hedgehog (Shh), leading to decreased levels of mature Shh ligand that is associated with the plasma membrane, and causing transient loss of Hh signaling, resulting in permanent FAS-related morphological abnormalities. In the present study, we further elucidate the mechanisms that regulate the intracellular transportation and secretion of Shh using the hepatic stellate cell line HSC8B. We have found that Shh is associated with caveolin-1 in the Golgi apparatus to form protein complexes and that these complexes are packaged as large punctuate structures (transport vesicles) that are transported to the plasma membrane in lipid raft microdomains. Alcohol exposure does not significantly interrupt translation of shh mRNA in endoplasmic reticulum (ER) or the trafficking of Shh from the ER to the Golgi apparatus. However, alcohol does prevent the entry of Shh into transport vesicles from the Golgi to the plasma membrane and specifically decreases the amount of caveolin-1/Shh complex found in lipid rafts, causing cytoplasmic accumulation of Shh and leading to a deficiency of Shh ligand secretion into the extracellular matrix.

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.

Histamine induces sonic hedgehog secretion across the apical membrane of polarized parietal cells

Objective:Sonic Hedgehog (Shh) is processed and secreted from parietal cells by a unique acid‐ and protease‐dependent mechanism (Zavros et al., 2007, JBC). The current study seeks to determine the cellular location of Shh within the gastric parietal cell.Methods:Shh expression was studied using cimetidine (resting) or histamine (stimulated)‐treated rabbit gastric glands and immunoisolated tubulovesicles. Apical (AP) and basolateral (BL) Shh secretion was studied using polarized human gastric cancer cells (23132/87).Results:Confocal microscopy of gastric glands triple stained for Shh, H+,K+‐ATPase and zymogen or neck cell markers showed expression of Shh within the canaliculus of stimulated parietal cells. Western blots showed Shh and H+,K+‐ATPase expression in resting immunoisolated tubulovesicles. While the Shh precursor was detected in resting membranes, increased expression of processed Shh was observed in stimulated tubulovesicles. Surface biotinylation of polarized 23132/87 cells revealed expression of the Shh precursor at the AP membrane and processed Shh secreted in the AP media. Shh was not detected in either the BL biotinylated membrane lysates or media.Conclusion:Shh is expressed in H+,K+‐ATPase‐containing membranes suggesting that processing and secretion occurs across the apical membrane of parietal cells.