Probes for INS

Hedgehog (HH) signaling participates in hepatobiliary repair after injury and is activated in patients with cholangiopathies. Cholangiopathies are associated with bile duct (BD) hyperplasia, including expansion of peribiliary glands, the niche for biliary progenitor cells. The inflammation‐associated cytokine interleukin (IL)‐33 is also up‐regulated in cholangiopathies, including cholangiocarcinoma. We hypothesized that HH signaling synergizes with IL‐33 in acute inflammation‐induced BD hyperplasia. We measured extrahepatic BD (EHBD) thickness and cell proliferation with and without an IL‐33 challenge in wild‐type mice, mice overexpressing Sonic HH (pCMV‐Shh), and mice with loss of the HH pathway effector glioma‐associated oncogene 1 (Gli1lacZ/lacZ). LacZ reporter mice were used to map the expression of HH effector genes in mouse EHBDs. An EHBD organoid (BDO) system was developed to study biliary progenitor cells in vitro. EHBDs from the HH overexpressing pCMV‐Shh mice showed increased epithelial cell proliferation and hyperplasia when challenged with IL‐33. In Gli1lacZ/lacZ mice, we observed a decreased proliferative response to IL‐33 and decreased expression of Il6. The HH ligands Shh and Indian HH (Ihh) were expressed in epithelial cells, whereas the transcriptional effectors Gli1, Gli2, and Gli3 and the HH receptor Patched1 (Ptch1) were expressed in stromal cells, as assessed by in situ hybridization and lacZ reporter mice. Although BDO cells lacked canonical HH signaling, they expressed the IL‐33 receptor suppression of tumorigenicity 2. Accordingly, IL‐33 treatment directly induced BDO cell proliferation in a nuclear factor κB‐dependent manner. Conclusion: HH ligand overexpression enhances EHBD epithelial cell proliferation induced by IL‐33. This proproliferative synergism of HH and IL‐33 involves crosstalk between HH ligand‐producing epithelial cells and HH‐responding stromal cells.

Genetic mutations in the Xylt1 gene are associated with Desbuquois dysplasia type II syndrome characterized by sever prenatal and postnatal short stature. However, the specific role of XylT-I in the growth plate is not completely understood. Here, we show that XylT-I is expressed and critical for the synthesis of proteoglycans in resting and proliferative but not in hypertrophic chondrocytes in the growth plate. We found that loss of XylT-I induces hypertrophic phenotype-like of chondrocytes associated with reduced interterritorial matrix. Mechanistically, deletion of XylT-I impairs the synthesis of long glycosaminoglycan chains leading to the formation of proteoglycans with shorter glycosaminoglycan chains. Histological and Second Harmonic Generation microscopy analysis revealed that deletion of XylT-I accelerated chondrocyte maturation and prevents chondrocytes columnar organization and arrangement in parallel of collagen fibers in the growth plate, suggesting that XylT-I controls chondrocyte maturation and matrix organization. Intriguingly, loss of XylT-I induced at embryonic stage E18.5 the migration of progenitor cells from the perichondrium next to the groove of Ranvier into the central part of epiphysis of E18.5 embryos. These cells characterized by higher expression of glycosaminoglycans exhibit circular organization then undergo hypertrophy and death creating a circular structure at the secondary ossification center location. Our study revealed an uncovered role of XylT-I in the synthesis of proteoglycans and provides evidence that the structure of glycosaminoglycan chains of proteoglycans controls chondrocyte maturation and matrix organization.

Usual Interstitial Pneumonia (UIP) is a histological pattern characteristic of Idiopathic Pulmonary Fibrosis (IPF). The UIP pattern is patchy with histologically normal lung adjacent to dense fibrotic tissue. At this interface, fibroblastic foci (FF) are present and are sites where myofibroblasts and extracellular matrix (ECM) accumulate. Utilizing laser capture microdissection coupled mass spectrometry (LCM-MS), we interrogated the FF, adjacent mature scar, and adjacent alveoli in 6 fibrotic (UIP/IPF) specimens plus 6 non-fibrotic alveolar specimens as controls. The data were subject to qualitative and quantitative analysis, and histologically validated. We found that the fibrotic alveoli protein signature is defined by immune deregulation as the strongest category. The fibrotic mature scar classified as end-stage fibrosis whereas the FF contained an overabundance of a distinctive ECM compared to non-fibrotic control. Furthermore, the FF is positive for both TGFB1 and TGFB3, whereas the aberrant basaloid cell lining of the FF is predominantly positive for TGFB2. In conclusion, spatial proteomics demonstrated distinct protein compositions in the histologically defined regions of UIP/IPF tissue. These data revealed that the FF is the main site of collagen biosynthesis and that the adjacent alveoli are abnormal. This new and essential information will inform future mechanistic studies on fibrosis progression.