Xingbin Ai

QSulf1 remodels the 6-O sulfation states of cell surface heparan sulfate proteoglycans to promote Wnt signaling

The 6-O sulfation states of cell surface heparan sulfate proteoglycans (HSPGs) are dynamically regulated to control the growth and specification of embryonic progenitor lineages. However, mechanisms for regulation of HSPG sulfation have been unknown. Here, we report on the biochemical and Wnt signaling activities of QSulf1, a novel cell surface sulfatase. Biochemical studies establish that QSulf1 is a heparan sulfate (HS) 6-O endosulfatase with preference, in particular, toward trisulfated IdoA2S-GlcNS6S disaccharide units within HS chains. In cells, QSulf1 can function cell autonomously to remodel the sulfation of cell surface HS and promote Wnt signaling when localized either on the cell surface or in the Golgi apparatus. QSulf1 6-O desulfation reduces XWnt binding to heparin and HS chains of Glypican1, whereas heparin binds with high affinity to XWnt8 and inhibits Wnt signaling. CHO cells mutant for HS biosynthesis are defective in Wnt-dependent Frizzled receptor activation, establishing that HS is required for Frizzled receptor function. Together, these findings suggest a two-state “catch or present” model for QSulf1 regulation of Wnt signaling in which QSulf1 removes 6-O sulfates from HS chains to promote the formation of low affinity HS–Wnt complexes that can functionally interact with Frizzled receptors to initiate Wnt signal transduction.

SULF1 and SULF2 regulate heparan sulfate-mediated GDNF signaling for esophageal innervation

Heparan sulfate (HS) plays an essential role in extracellular signaling during development. Biochemical studies have established that HS binding to ligands and receptors is regulated by the fine 6-O-sulfated structure of HS; however, mechanisms that control sulfated HS structure and associated signaling functions in vivo are not known. Extracellular HS 6-O-endosulfatases, SULF1 and SULF2, are candidate enzymatic regulators of HS 6-O-sulfated structure and modulate HS-dependent signaling. To investigate Sulf regulation of developmental signaling, we have disrupted Sulf genes in mouse and identified redundant functions of Sulfs in GDNF-dependent neural innervation and enteric glial formation in the esophagus, resulting in esophageal contractile malfunction in Sulf1-/-;Sulf2-/- mice. SULF1 is expressed in GDNF-expressing esophageal muscle and SULF2 in innervating neurons, establishing their direct functions in esophageal innervation. Biochemical and cell signaling studies show that Sulfs are the major regulators of HS 6-O-desulfation, acting to reduce GDNF binding to HS and to enhance GDNF signaling and neurite sprouting in the embryonic esophagus. The functional specificity of Sulfs in GDNF signaling during esophageal innervation was established by showing that the neurite sprouting is selectively dependent on GDNF, but not on neurotrophins or other signaling ligands. These findings provide the first in vivo evidence that Sulfs are essential developmental regulators of cellular HS 6-O-sulfation for matrix transmission and reception of GDNF signal from muscle to innervating neurons.

Substrate Specificity and Domain Functions of Extracellular Heparan Sulfate 6-O-Endosulfatases, QSulf1 and QSulf2

The extracellular sulfatases (Sulfs) are an evolutionally conserved family of heparan sulfate (HS)-specific 6-O-endosulfatases. These enzymes remodel the 6-O-sulfation of cell surface HS chains to promote Wnt signaling and inhibit growth factor signaling for embryonic tissue patterning and control of tumor growth. In this study we demonstrate that the avian HS endosulfatases, QSulf1 and QSulf2, exhibit the same substrate specificity toward a subset of trisulfated disaccharides internal to HS chains. Further, we show that both QSulfs associate exclusively with cell membrane and are enzymatically active on the cell surface to desulfate both cell surface and cell matrix HS. Mutagenesis studies reveal that conserved amino acid regions in the hydrophilic domains of QSulf1 and QSulf2 have multiple functions, to anchor Sulf to the cell surface, bind to HS substrates, and to mediate HS 6-O-endosulfatase enzymatic activity. Results of our current studies establish the hydrophilic domain (HD) of Sulf enzymes as an essential multifunctional domain for their unique endosulfatase activities and also demonstrate the extracellular activity of Sulfs for desulfation of cell surface and cell matrix HS in the control of extracellular signaling for embryonic development and tumor progression.

Ventral Neural Progenitors Switch toward an Oligodendroglial Fate in Response to Increased Sonic Hedgehog (Shh) Activity: Involvement of Sulfatase 1 in Modulating Shh Signaling in the Ventral Spinal Cord

In the embryonic chick ventral spinal cord, the initial emergence of oligodendrocytes is a relatively late event that depends on prolonged Sonic hedgehog (Shh) signaling. In this report, we show that specification of oligodendrocyte precursors (OLPs) from ventral Nkx2.2-expressing neural progenitors occurs precisely when these progenitors stop generating neurons, indicating that the mechanism of the neuronal/oligodendroglial switch is a common feature of ventral OLP specification. We further show that an experimental early increase in the concentration of Shh is sufficient to induce premature specification of OLPs at the expense of neuronal genesis indicating that the relative doses of Shh received by ventral progenitors determine whether they become neurons or glia. Accordingly, we observe that the Shh protein accumulates at the apical surface of Nkx2.2-expressing cells just before OLP specification, providing direct evidence that these cells are subjected to a higher concentration of the morphogen when they switch to an oligodendroglial fate. Finally, we show that this abrupt change in Shh distribution is most likely attributable to the timely activity of Sulfatase 1 (Sulf1), a secreted enzym that modulates the sulfation state of heparan sulfate proteoglycans. Sulf1 is expressed in the ventral neuroepithelium just before OLP specification, and we show that its experimental overexpression leads to apical concentration of Shh on neuroepithelial cells, a decisive event for the switch of ventral neural progenitors toward an oligodendroglial fate.

Derivation of lung mesenchymal lineages from the fetal mesothelium requires hedgehog signaling for mesothelial cell entry

Recent studies have shown that mesothelial progenitors contribute to mesenchymal lineages of developing organs. To what extent the overlying mesothelium contributes to lung development remains unknown. To rigorously address this question, we employed Wt1CreERT2/+ mice for high-fidelity lineage tracing after confirming that Cre recombinase was mesothelial specific and faithfully recapitulated endogenous Wilms’ tumor 1 (Wt1) gene expression. We visualized WT1+ mesothelial cell entry into the lung by live imaging and identified their progenies in subpopulations of bronchial smooth muscle cells, vascular smooth muscle cells and desmin+ fibroblasts by lineage tagging. Derivation of these lineages was only observed with Cre recombinase activation during early lung development. Using loss-of-function assays in organ cultures, and targeted mesothelial-restricted hedgehog loss-of-function mice, we demonstrated that mesothelial cell movement into the lung requires the direct action of hedgehog signaling. By contrast, hedgehog signaling was not required for fetal mesothelial heart entry. These findings further support a paradigm wherein the mesothelium is a source of progenitors for mesenchymal lineages during organogenesis and indicate that signals controlling mesothelial cell entry are organ specific.

A Shh/miR-206/BDNF cascade coordinates innervation and formation of airway smooth muscle.

Dysfunctional neural control of airway smooth muscle (ASM) is involved in inflammatory diseases, such as asthma. However, neurogenesis in the lung is poorly understood. This study uses mouse models to investigate developmental mechanisms of ASM innervation, a process that is highly coordinated with ASM formation during lung branching morphogenesis. We show that brain-derived neurotrophic factor (BDNF) is an essential ASM-derived signal for innervation. Although BDNF mRNA expression is temporally dissociated with ASM formation and innervation, BDNF protein is coordinately produced through post-transcriptional suppression by miR-206. Using a combination of chemical and genetic approaches to modulate sonic hedgehog (Shh) signaling, a pathway essential for lung branching and ASM formation, we show that Shh signaling blocks miR-206 expression, which in turn increases BDNF protein expression. Together, our work uncovers a functional cascade that involves Shh, miR-206 and BDNF to coordinate ASM formation and innervation.

Molecular determinants of lung development.

Development of the pulmonary system is essential for terrestrial life. The molecular pathways that regulate this complex process are beginning to be defined, and such knowledge is critical to our understanding of congenital and acquired lung diseases. A recent workshop was convened by the National Heart, Lung, and Blood Institute to discuss the developmental principles that regulate the formation of the pulmonary system. Emerging evidence suggests that key developmental pathways not only regulate proper formation of the pulmonary system but are also reactivated upon postnatal injury and repair and in the pathogenesis of human lung diseases. Molecular understanding of early lung development has also led to new advances in areas such as generation of lung epithelium from pluripotent stem cells. The workshop was organized into four different topics, including early lung cell fate and morphogenesis, mechanisms of lung cell differentiation, tissue interactions in lung development, and environmental impact on early lung development. Critical points were raised, including the importance of epigenetic regulation of lung gene expression, the dearth of knowledge on important mesenchymal lineages within the lung, and the interaction between the developing pulmonary and cardiovascular system. This manuscript describes the summary of the discussion along with general recommendations to overcome the gaps in knowledge in lung developmental biology.

Heparan Sulfate 6-O-endosulfatases (Sulfs) Coordinate the Wnt Signaling Pathways to Regulate Myoblast Fusion during Skeletal Muscle Regeneration

Background: The Sulf enzymes regulate HS-dependent extracellular signaling during skeletal muscle regeneration. Results: The skeletal muscle-specific Sulf double null mice exhibit reduced new myofiber size and defective Wnt signaling. Conclusion: Sulfs promote canonical Wnt signaling to antagonize noncanonical signaling, thereby enhancing myoblast fusion. Significance: Understanding the mechanisms of myoblast fusion will help to enhance the efficacy of muscle regeneration. Skeletal muscle regeneration is mediated by satellite cells (SCs). Upon injury, SCs undergo self-renewal, proliferation, and differentiation into myoblasts followed by myoblast fusion to form new myofibers. We previously showed that the heparan sulfate (HS) 6-O-endosulfatases (Sulf1 and -2) repress FGF signaling to induce SC differentiation during muscle regeneration. Here, we identify a novel role of Sulfs in myoblast fusion using a skeletal muscle-specific Sulf double null (SulfSK-DN) mouse. Regenerating SulfSK-DN muscles exhibit reduced canonical Wnt signaling and elevated non-canonical Wnt signaling. In addition, we show that Sulfs are required to repress non-canonical Wnt signaling to promote myoblast fusion. Notably, skeletal muscle-relevant non-canonical Wnt ligands lack HS binding capacity, suggesting that Sulfs indirectly repress this pathway. Mechanistically, we show that Sulfs reduce the canonical Wnt-HS binding and regulate colocalization of the co-receptor LRP5 with caveolin3. Therefore, Sulfs may increase the bioavailability of canonical Wnts for Frizzled receptor and LRP5/6 interaction in lipid raft, which may in turn antagonize non-canonical Wnt signaling. Furthermore, changes in subcellular distribution of active focal adhesion kinase (FAK) are associated with the fusion defect of Sulf-deficient myoblasts and upon non-canonical Wnt treatment. Together, our findings uncover a critical role of Sulfs in myoblast fusion by promoting antagonizing canonical Wnt signaling activities against the noncanonical Wnt pathway during skeletal muscle regeneration.

A subtractive approach to characterize genes with regionalized expression in the gliogenic ventral neuroepithelium: identification of chick sulfatase 1 as a new oligodendrocyte lineage gene.

To address the question of the origin of glial cells and the mechanisms leading to their specification, we have sought to identify novel genes expressed in glial progenitors. We adopted suppression subtractive hybridization (SSH) to establish a chick cDNA library enriched for genes specifically expressed at 6 days of incubation (E6) in the ventral neuroepithelium, a tissue previously shown to contain glial progenitors. Screens were then undertaken to select differentially expressed cDNAs, and out of 82 unique SSH clones, 21 were confirmed to display a regionalized expression along the dorsoventral axis of the E6 ventral neuroepithelium. Among these, we identified a transcript coding for the chick orthologue of Sulf1, a recently identified cell surface sulfatase, as a new, early marker of oligodendrocyte (OL) precursors in the chick embryonic spinal cord. This study provides groundwork for the further identification of genes involved in glial specification.

Aspirin-triggered resolvin D1 is produced during self-resolving gram-negative bacterial pneumonia and regulates host immune responses for the resolution of lung inflammation.

Bacterial pneumonia is a leading cause of morbidity and mortality worldwide. Host responses to contain infection and mitigate pathogen-mediated lung inflammation are critical for pneumonia resolution. Aspirin-triggered resolvin D1 (AT-RvD1; 7S,8R,17R-trihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid) is a lipid mediator (LM) that displays organ-protective actions in sterile lung inflammation, and regulates pathogen-initiated cellular responses. Here, in a self-resolving murine model of Escherichia coli pneumonia, LM metabololipidomics performed on lungs obtained at baseline, 24, and 72 h after infection uncovered temporal regulation of endogenous AT-RvD1 production. Early treatment with exogenous AT-RvD1 (1 h post infection) enhanced clearance of E. coli and Pseudomonas aeruginosa in vivo, and lung macrophage phagocytosis of fluorescent bacterial particles ex vivo. Characterization of macrophage subsets in the alveolar compartment during pneumonia identified efferocytosis by infiltrating macrophages (CD11bHi CD11cLow) and exudative macrophages (CD11bHi CD11cHi). AT-RvD1 increased efferocytosis by these cells ex vivo, and accelerated neutrophil clearance during pneumonia in vivo. These anti-bacterial and pro-resolving actions of AT-RvD1 were additive to antibiotic therapy. Taken together, these findings suggest that the pro-resolving actions of AT-RvD1 during pneumonia represent a novel host-directed therapeutic strategy to complement the current antibiotic-centered approach for combatting infections.