Pawel Niewiadomski

Interaction of PACAP with Sonic hedgehog reveals complex regulation of the Hedgehog pathway by PKA

Sonic hedgehog (Shh) signaling is essential for proliferation of cerebellar granule cell progenitors (cGCPs) and its aberrant activation causes a cerebellar cancer medulloblastoma. Pituitary adenylate cyclase activating polypeptide (PACAP) inhibits Shh-driven proliferation of cGCPs and acts as tumor suppressor in murine medulloblastoma. We show that PACAP blocks canonical Shh signaling by a mechanism that involves activation of protein kinase A (PKA) and inhibition of the translocation of the Shh-dependent transcription factor Gli2 into the primary cilium. PKA is shown to play an essential role in inhibiting gene transcription in the absence of Shh, but global PKA activity levels are found to be a poor predictor of the degree of Shh pathway activation. We propose that the core Shh pathway regulates a small compartmentalized pool of PKA in the vicinity of primary cilia. GPCRs that affect global PKA activity levels, such as the PACAP receptor, cooperate with the canonical Shh signal to regulate Gli protein phosphorylation by PKA. This interaction serves to fine-tune the transcriptional and physiological function of the Shh pathway.

Negative Regulation of Hedgehog Signaling by Liver X Receptors

Hedgehog (Hh) signaling is indispensable in embryonic development, and its dysregulated activity results in severe developmental disorders as shown by genetic models of naturally occurring mutations in animal and human pathologies. Hh signaling also functions in postembryonic development and adult tissue homeostasis, and its aberrant activity causes various human cancers. Better understanding of molecular regulators of Hh signaling is of fundamental importance in finding new strategies for pathway modulation. Here, we identify liver X receptors (LXRs), members of the nuclear hormone receptor family, as previously unrecognized negative regulators of Hh signaling. Activation of LXR by specific pharmacological ligands, TO901317 and GW3965, inhibited the responses of pluripotent bone marrow stromal cells and calvaria organ cultures to sonic Hh, resulting in the inhibition of expression of Hh-target genes, Gli1 and Patched1, and Gli-dependent transcriptional activity. Moreover, LXR ligands inhibited sonic Hh-induced differentiation of bone marrow stromal cells into osteoblasts. Elimination of LXRs by small interfering RNA inhibited ligand-induced inhibition of Hh target gene expression. Furthermore, LXR ligand did not inhibit Hh responsiveness in mouse embryonic fibroblasts that do not express LXRs, whereas introduction of LXR into these cells reestablished the inhibitory effects. Daily oral administration of TO901317 to mice after 3 d significantly inhibited baseline Hh target-gene expression in liver, lung, and spleen. Given the importance of modulating Hh signaling in various physiological and pathological settings, our findings suggest that pharmacological targeting of LXRs may be a novel strategy for Hh pathway modulation.

Pituitary adenylyl cyclase activating polypeptide inhibits gli1 gene expression and proliferation in primary medulloblastoma derived tumorsphere cultures

BackgroundHedgehog (HH) signaling is critical for the expansion of granule neuron precursors (GNPs) within the external granular layer (EGL) during cerebellar development. Aberrant HH signaling within GNPs is thought to give rise to medulloblastoma (MB) - the most commonly-observed form of malignant pediatric brain tumor. Evidence in both invertebrates and vertebrates indicates that cyclic AMP-dependent protein kinase A (PKA) antagonizes HH signalling. Receptors specific for the neuropeptide pituitary adenylyl cyclase activating polypeptide (PACAP, gene name ADCYAP1) are expressed in GNPs. PACAP has been shown to protect GNPs from apoptosis in vitro, and to interact with HH signaling to regulate GNP proliferation. PACAP/ptch1 double mutant mice exhibit an increased incidence of MB compared to ptch1 mice, indicating that PACAP may regulate HH pathway-mediated MB pathogenesis.MethodsPrimary MB tumorsphere cultures were prepared from thirteen ptch1+/-/p53+/- double mutant mice and treated with the smoothened (SMO) agonist purmorphamine, the SMO antagonist SANT-1, the neuropeptide PACAP, the PKA activator forskolin, and the PKA inhibitor H89. Gene expression of gli1 and [3H]-thymidine incorporation were assessed to determine drug effects on HH pathway activity and proliferation, respectively. PKA activity was determined in cell extracts by Western blotting using a phospho-PKA substrate antibody.ResultsPrimary tumor cells cultured for 1-week under serum-free conditions grew as tumorspheres and were found to express PAC1 receptor transcripts. Gli1 gene expression was significantly reduced by SANT-1, PACAP and forskolin, but was unaffected by purmorphamine. The attenuation of gli1 gene expression by PACAP was reversed by the PKA inhibitor H89, which also blocked PKA activation. Treatment of tumorsphere cultures with PACAP, forskolin, and SANT-1 for 24 or 48 hours reduced proliferation.ConclusionsPrimary tumorspheres derived from ptch1+/-/p53+/- mice exhibit constitutive HH pathway activity. PACAP antagonizes HH signalling in these cells in a manner blocked by the PKA antagonist H89. PACAP and pharmacological activation of PKA also inhibited proliferation. Our data suggests that regulation of HH signaling by PACAP/PKA signaling may provide an alternative to SMO inhibition for the treatment of MB.

Involvement of unconventional myosin VI in myoblast function and myotube formation

The important role of unconventional myosin VI (MVI) in skeletal and cardiac muscle has been recently postulated (Karolczak et al. in Histochem Cell Biol 139:873–885, 2013). Here, we addressed for the first time a role for this unique myosin motor in myogenic cells as well as during their differentiation into myotubes. During myoblast differentiation, the isoform expression pattern of MVI and its subcellular localization underwent changes. In undifferentiated myoblasts, MVI-stained puncti were seen throughout the cytoplasm and were in close proximity to actin filaments, Golgi apparatus, vinculin-, and talin-rich focal adhesion as well as endoplasmic reticulum. Colocalization of MVI with endoplasmic reticulum was enhanced during myotube formation, and differentiation-dependent association was also seen in sarcoplasmic reticulum of neonatal rat cardiomyocytes (NRCs). Moreover, we observed enrichment of MVI in myotube regions containing acetylcholine receptor-rich clusters, suggesting its involvement in the organization of the muscle postsynaptic machinery. Overexpression of the H246R MVI mutant (associated with hypertrophic cardiomyopathy) in myoblasts and NRCs caused the formation of abnormally large intracellular vesicles. MVI knockdown caused changes in myoblast morphology and inhibition of their migration. On the subcellular level, MVI-depleted myoblasts exhibited aberrations in the organization of actin cytoskeleton and adhesive structures as well as in integrity of Golgi apparatus and endoplasmic reticulum. Also, MVI depletion or overexpression of H246R mutant caused the formation of significantly wider or aberrant myotubes, respectively, indicative of involvement of MVI in myoblast differentiation. The presented results suggest an important role for MVI in myogenic cells and possibly in myoblast differentiation.

Selective targeting of HDAC1/2 elicits anticancer effects through Gli1 acetylation in preclinical models of SHH Medulloblastoma

SHH Medulloblastoma (SHH-MB) is a pediatric brain tumor characterized by an inappropriate activation of the developmental Hedgehog (Hh) signaling. SHH-MB patients treated with the FDA-approved vismodegib, an Hh inhibitor that targets the transmembrane activator Smoothened (Smo), have shown the rapid development of drug resistance and tumor relapse due to novel Smo mutations. Moreover, a subset of patients did not respond to vismodegib because mutations were localized downstream of Smo. Thus, targeting downstream Hh components is now considered a preferable approach. We show here that selective inhibition of the downstream Hh effectors HDAC1 and HDAC2 robustly counteracts SHH-MB growth in mouse models. These two deacetylases are upregulated in tumor and their knockdown inhibits Hh signaling and decreases tumor growth. We demonstrate that mocetinostat (MGCD0103), a selective HDAC1/HDAC2 inhibitor, is a potent Hh inhibitor and that its effect is linked to Gli1 acetylation at K518. Of note, we demonstrate that administration of mocetinostat to mouse models of SHH-MB drastically reduces tumor growth, by reducing proliferation and increasing apoptosis of tumor cells and prolongs mouse survival rate. Collectively, these data demonstrate the preclinical efficacy of targeting the downstream HDAC1/2-Gli1 acetylation in the treatment of SHH-MB.

Pituitary adenylyl cyclase-activating peptide counteracts hedgehog-dependent motor neuron production in mouse embryonic stem cell cultures.

Pituitary adenylyl cyclase‐activating peptide (PACAP; ADCYAP1) is a neuropeptide that regulates a wide array of functions within the brain and periphery. We and others have previously demonstrated that PACAP and its high‐affinity receptor PAC1 are expressed in the embryonic mouse neural tube, suggesting that PACAP plays a role in early brain development. Moreover, we previously showed that PACAP antagonizes the mitotic action of Sonic hedgehog (Shh) in postnatal cerebellar granule precursors. In the present study, we demonstrate that PACAP completely blocked Shh‐dependent motor neuron generation from embryonic stem cell cultures and reduced mRNA levels of the Shh target gene Gli‐1 and several ventral spinal cord patterning genes. In vivo examination of motor neuron and other patterning markers in embryonic day 12.5 spinal cords of wild‐type and PACAP‐deficient mice by immunofluorescence, on the other hand, revealed no obvious alterations in expressions of Islet1/2, MNR2, Lim1/2, Nkx2.2, or Shh, although the Pax6‐positive area was slightly expanded in PACAP‐deficient spinal cord. Caspase‐3 staining revealed low, and similar, numbers of cells undergoing apoptosis in embryonic wild‐type vs. PACAP‐deficient spinal cords, whereas a slight but significant increase in number of mitotic cells was observed in PACAP‐deficient mice. Thus, although PACAP has a strong capacity to counteract Shh signaling and motor neuron production in vitro, corresponding patterning defects associated with PACAP loss may be obscured by compensatory mechanisms.

Neurotransmitter and Immunomodulatory Actions of VIP and PACAP: Lessons from Knockout Mice

Vasoactive Intestinal Peptide (VIP) and Pituitary Adenylyl Cyclase Activating Peptide (PACAP) are two closely related neuropeptides in the secretin family. They are widely expressed in the central and peripheral nervous systems, where they are classically thought to act as neurotransmitters or neuromodulators. They interact with high affinity receptors to regulate numerous behaviors as well as gastrointestinal, endocrine, cardiopulmonary, reproductive and immune functions. The recent generation of mice that specifically lack or overexpress VIP, PACAP or their receptors has yielded much new knowledge and enabled investigators to better understand the biological roles of these peptides and their impact on health. In this review, we attempt to summarize the major findings, but focus in greatest detail on the circadian and immune functions.

Liprin-α-1 is a novel component of the murine neuromuscular junction and is involved in the organization of the postsynaptic machinery

Neuromuscular junctions (NMJs) are specialized synapses that connect motor neurons to skeletal muscle fibers and orchestrate proper signal transmission from the nervous system to muscles. The efficient formation and maintenance of the postsynaptic machinery that contains acetylcholine receptors (AChR) are indispensable for proper NMJ function. Abnormalities in the organization of synaptic components often cause severe neuromuscular disorders, such as muscular dystrophy. The dystrophin-associated glycoprotein complex (DGC) was shown to play an important role in NMJ development. We recently identified liprin-α-1 as a novel binding partner for one of the cytoplasmic DGC components, α-dystrobrevin-1. In the present study, we performed a detailed analysis of localization and function of liprin-α-1 at the murine NMJ. We showed that liprin-α-1 localizes to both pre- and postsynaptic compartments at the NMJ, and its synaptic enrichment depends on the presence of the nerve. Using cultured muscle cells, we found that liprin-α-1 plays an important role in AChR clustering and the organization of cortical microtubules. Our studies provide novel insights into the function of liprin-α-1 at vertebrate neuromuscular synapses.

PACAP Promotes Matrix-Driven Adhesion of Cultured Adult Murine Neural Progenitors

New neurons are born throughout the life of mammals in germinal zones of the brain known as neurogenic niches: the subventricular zone of the lateral ventricles and the subgranular zone of the dentate gyrus of the hippocampus. These niches contain a subpopulation of cells known as adult neural progenitor cells (aNPCs), which self-renew and give rise to new neurons and glia. aNPCs are regulated by many factors present in the niche, including the extracellular matrix (ECM). We show that the neuropeptide PACAP (pituitary adenylate cyclase-activating polypeptide) affects subventricular zone-derived aNPCs by increasing their surface adhesion. Gene array and reconstitution assays indicate that this effect can be attributed to the regulation of ECM components and ECM-modifying enzymes in aNPCs by PACAP. Our work suggests that PACAP regulates a bidirectional interaction between the aNPCs and their niche: PACAP modifies ECM production and remodeling, in turn the ECM regulates progenitor cell adherence. We speculate that PACAP may in this manner help restrict adult neural progenitors to the stem cell niche in vivo, with potential significance for aNPC function in physiological and pathological states.

Measuring Expression Levels of Endogenous Gli Genes by Immunoblotting and Real-Time PCR

Gli proteins are transcription factors that mediate the transcriptional effects of Hedgehog signaling in vertebrates. The activities of Gli2 and Gli3 are regulated primarily by posttranslational modifications, while Gli1 is mostly regulated at the transcriptional level. Detection of endogenous Gli proteins had been hampered by lack of good antibodies, but this problem has been mostly resolved in recent years. In this chapter we describe methods of detecting expression of endogenous Gli genes in whole-cell lysates and in subcellular fractions and also provide protocols for the measurement of Gli mRNA levels by quantitative real-time reverse transcriptase PCR (qPCR).