Low-density lipoprotein receptor-related protein 1 (LRP1) is a multifunctional endocytic receptor whose dysfunction is linked to developmental dysplasia of the hip, osteoporosis and osteoarthritis. Our work addresses the critical question of how these skeletal pathologies emerge. Here, we show the abundant expression of LRP1 in skeletal progenitor cells at mouse embryonic stage E10.5 and onwards, especially in the perichondrium, the stem cell layer surrounding developing limbs essential for bone formation. Lrp1 deficiency in these stem cells causes joint fusion, malformation of cartilage/bone template and markedly delayed or lack of primary ossification. These abnormalities, which resemble phenotypes associated with Wnt signalling pathways, result in severe and persistent skeletal defects including a severe deficit in hip joint and patella, and markedly deformed and low-density long bones leading to dwarfism and impaired mobility. Mechanistically, we show that LRP1 regulates core non-canonical Wnt/planar cell polarity (PCP) components that may explain the malformation of long bones. LRP1 directly binds to Wnt5a, facilitates its cell-association and endocytic degradation and recycling. In the developing limbs, LRP1 partially colocalises with Wnt5a and its deficiency alters abundance and distribution of Wnt5a and Vangl2. Finally, using Xenopus as a model system, we show the regulatory role for LRP1 in Wnt/PCP signalling. We propose that in skeletal progenitors, LRP1 plays a critical role in formation and maturity of multiple bones and joints by regulating Wnt signalling, providing novel insights into the fundamental processes of morphogenesis and the emergence of skeletal pathologies.

Background: Articular soft tissue mineralization and ossification are clear pathological signs of osteoarthritis (OA) joints. However their molecular and cellular aetiologies remain largely unknown. Transforming growth factor beta (TGF-3) family members are known contributors to both pathological ossification and osteoarthritis development. In this study, we used a fibrillin-1 (Fbn1) mutant mouse, the tight skin (TSK) mouse, to define the detrimental effects of abnormal Fbn1 in TSK mice and known high TGF-3 activity in joint pathology such as articular soft tissue mineralization and ossification. Methods: Knee joints of male and female TSK and wild-type (WT) littermates were analysed by micro-computed tomography (micro-CT) imaging and histology for articular soft tissue pathologies, as well as OA severity. Both aged (10, 26, 35 and 52 weeks) and following in vivo non-invasive repetitive joint overloading were used. Results: We find that male TSK mice develop spontaneous soft tissue ossification from 26 weeks of age, followed by increased osteoarthritis at 1 year-old. In addition, knee joint overloading induced ligament and meniscal mineralisation and ossification in both WT and TSK male mice, but were significantly more severe in TSK knees, including ossification of the patella ligament and synovial lining. In contrast, female TSK knees did not develop more severe soft tissue mineralisation compared to littermate WT mice in neither aged nor overloaded knees. Conclusions: We conclude that Fbn1 mutation, and possibly overactive TGF-3 activity in TSK mice, induce articular soft tissue ossification and osteoarthritis in a sex-specific manner. Further studies are needed to confirm the specific signalling involved and the relative protection from female mice from such pathologies.

CCN1 +/+ stroma vasculature microct

CCN1 expression by fibroblasts is associated with increased tumor penetration of CD4+ T cells and resistance to anti-PD1 checkpoint inhibitors. A, Tumors were subjected to flow cytometry with anti-CD4 anti-CD8 antibodies. Percentage of positive CD45-staining cells are indicated wild type (n = 3, CCN1f/f) and CCN1-deficient (n = 3, CCN1−/−) tumors (Student t test). Primary flow data of the median value is presented Tissue was examined after 14 days of tumor growth. B, Boxplot shows higher levels of CAF-specific CCN1 scores in patients on checkpoint inhibitors with progressive disease (GSE78220). A six-gene set of CAF-specific genes correlating with CCN1 (PDGFRA, COL1A1, DCN, TAGLN, COL6A3, and LPAR1), as described in Materials and Methods, was analyzed. P value (unpaired t test) displayed in top left corner. n = 14 in the responders and n = 12 in the progressive disease. C and D, Loss of CCN1 expression results in reduced MMP-9 expression in CCN1-deficient mice. Real-time PCR analysis of RNAs isolated from Ccn1+/+ and Ccn1−/− dermal fibroblasts from 3 different mice (N = 3; C) or skin (N = 4 Ccn1+/+, N = 8, Ccn1−/−; D) reveal that CCN1-deficient fibroblasts have reduced expression of the anti-PD1 resistance marker/effector MMP-9 (Student unpaired t test; *, P < 0.05). Please note that D was conducted on identical skin samples to those published previously (29).

CCN1 deficient stroma vasculature microct

Mice harboring Col1A2-Cre-fibroblast–specific deletion for Ccn1 show impaired metastasis of B16F10 melanoma cells to the lung. A–C, Mice were examined after 14 days of tumor growth. A, Sections of tumor stroma were stained with anti-CCN1 antibody (N = 3, representative images are shown), verifying loss of CCN1 protein expression in mice deleted for CCN1 in fibroblasts. Note CCN1 staining remains in the tumor. Scale bar = 50 µm. B, Representative images of lung sections from wild-type or mice harboring a deletion for Ccn1 in Col1A2-Cre-fibroblasts. Hematoxylin and eosin was used to reveal detect dense metastatic foci (purple). Scale Bar = 300 µm. Total area of the lung section covered by metastases was quantified. Deletion of Ccn1 caused reduced metastasis (N = 6, ***, P < 0.001). C, Reduced CCN2 expression in the stroma did not significantly alter tumor growth. Kruskal–Wallis analysis (N = 8). D, To more accurately define the cells in which CCN1 was deleted, three-week old Col1A2-cre(ER)T/); mTmG mice were injected for 5 consecutive days with tamoxifen to activate Cre recombinase. Six weeks later, skin was isolated, digested with collagenase, and cells expressing GFP were isolated by FACS, and subjected to RNA-seq analysis. Col1A2 lineage fibroblasts were identified as universal fibroblasts based on the gene expression of Pi16, Col15a1, C3, and Cd84 genes, but not papillary or reticular fibroblasts based on the gene expression of Crabp1 and Defb8 or Nexn, Trim63, Actn2, and Hspb7, respectively.

Purpose (the aim of the study): Wnt signalling is an evolutionarily conserved pathway that is critical in cell fate determination, organ development and post-natal tissue homeostasis. Both canonical and non-canonical Wnt/planar cell polarity (PCP) pathways are implicated in key stages of limb development. Unbalanced Wnt signalling leads to the development of osteoarthritis (OA). Our recent study showed that key components of the Wnt/PCP signalling pathway, namely Wnt5a and Wnt11, bind to the low-density lipoprotein receptor-related protein 1 (LRP1). LRP1 is a widely expressed cell-surface receptor that mediates clathrin-dependent endocytosis of a diverse array of ligands. In this study, we have investigated the endocytic regulation of Wnt5a by LRP1 and the significance of their interaction using Xenopus embryonic development and transgenic mouse models.

BackgroundIncreased homogentisic acid (HGA) in alkaptonuria (AKU) causes severe arthritis. Nitisinone reduces the production of HGA, but whether it also decreases arthroplasty was examined in 237 AKU patients.Patients and methodsPatients attending the United Kingdom National Alkaptonuria Centre (NAC) and the Suitability of Nitisinone in Alkaptonuria 2 (SONIA 2) study were studied. Assessments included questionnaires eliciting details of arthroplasty. Nitisinone was administered from baseline, 2 mg in the NAC and 10 mg in SONIA 2. In SONIA 2, subgroups consisted of those with baseline arthroplasty on and not on nitisinone (BR + N+, BR + N-), as well as those without baseline arthroplasty on and not on nitisinone (BR-N+, BR-N-).ResultsIn the SONIA2 subgroups, new joint replacement (JR) probabilities after baseline were significantly different (BR + N+, BR + N-, BR-N+, BR-N-) (χ2 = 23.3, p < 0.001); mean (SD) was 3.8 (0.1) years in BR-N-, 3.7 (0.1) years in BR-N+, 3.4 (0.3) years in BR + N-, and 3.0 (0.3) years in BR + N+. Further, the BR + N- showed more JR than the BR-N- subgroup (p < 0.01), while BR + N+ similarly showed more JR than the BR-N+ subgroup (p < 0.001).In the NAC, the BR- group had a mean age of 51.6 (7.0) years at baseline but 57.7 (8.7) years at final follow up during nitisinone therapy and showed only 7 incident JR. The BR+ group had an age at baseline of 57.4 (8.5) years and had undergone 94 JRs at baseline.ConclusionThe incidence of arthroplasty was earlier and more frequent after the first JR and was not affected by nitisinone.

Deletion of Ccn1 in Col1A2-Cre-fibroblasts reduces tumor vasculature. A, Tumors in wild-type or mice deleted for Ccn1 in Col1A2-Cre-fibroblasts were perfused with a CT-contrast agent, as described in Materials and Methods. Volume (mL) of tumor occupied by vasculature was calculated. Mice lacking Ccn1 in their fibroblasts had significantly reduced vascular volume (t test, n = 3, P < 0.05). B, Tumors and associated stroma in wild-type or mice deleted for Ccn1 in fibroblasts were stained with anti-CD31 antibodies (t test; n = 3; *, P < 0.05).

CCN1 deficient stroma vasculature microct

Multivariate analysis of CAF-specific CCN1 score as a prognostic marker for reduced overall survival in GSE78220 (36) in patients on anti-PD1 checkpoint inhibitors

Loss of CCN1 expression from CAFs does not affect cell proliferation or apoptosis in a syngeneic model of melanoma metastasis. A, PCNA assay in (top) tumor and (bottom) stroma. Cell proliferation was detected using an anti-PCNA antibody. Representative images shown; graphs show the mean percentage of PCNA positive cells in CAFs ± SEM; CCN1f/fn = 7 and CCN1−/−n = 4, Student t test. B, TUNEL assay to detect apoptosis in stroma. Representative images shown; graphs show the mean percentage of TUNEL positive cells in stroma ± SEM, CCN1f/fn = 7 and CCN1−/−n = 4, Student t test.

Abstract Melanoma is the leading cause of skin cancer–related death. As prognosis of patients with melanoma remains problematic, identification of new therapeutic targets remains essential. Matricellular proteins are nonstructural extracellular matrix proteins. They are secreted into the tumor microenvironment to coordinate behavior among different cell types, yet their contribution to melanoma is underinvestigated. Examples of matricellular proteins include those comprising the CCN family. The CCN family member, CCN1, is highly proangiogenic. Herein, we show that, in human patients with melanoma, although found in several tumor cell types, CCN1 is highly expressed by a subset of cancer-associated fibroblasts (CAF) in patients with melanoma and this expression correlates positively with expression of proangiogenic genes and progressive disease/resistance to anti-PD1 checkpoint inhibitors. Consistent with these observations, in a syngeneic C57BL6 mouse model of melanoma, loss of CCN1 expression from Col1A2-Cre-, herein identified as “universal,” fibroblasts, impaired metastasis of subcutaneously injected B16F10 tumor cells to lung, concomitant with disrupted neovascularization and collagen organization. Disruption of the extracellular matrix in the loss of CCN1 was validated using a novel artificial intelligence–based image analysis platform that revealed significantly decreased phenotypic fibrosis and composite morphometric collagen scores. As drug resistance is linked to matrix deposition and neoangiogenesis, these data suggest that CCN1, due to its multifaceted role, may represent a novel therapeutic target for drug-resistant melanoma. Our data further emphasize the essential role that cancer-associated, (universal) Col1A2-Cre-fibroblasts and extracellular matrix remodeling play in coordinating behavior among different cell types within the tumor microenvironment. Significance: In human patients, the expression of proangiogenic matricellular protein CCN1 in CAFs correlates positively with expression of stroma and angiogenic markers and progressive disease/resistance to checkpoint inhibitor therapy. In an animal model, loss of CCN1 from CAFs impaired metastasis of melanoma cells, neovascularization, and collagen deposition, emphasizing that CAFs coordinate cellular behavior in a tumor microenvironment and that CCN1 may be a novel target.

Altered activity of specific enzymes in phenylalanine-tyrosine (phe-tyr) metabolism results in incomplete breakdown of various metabolite substrates in this pathway. Increased biofluid concentration and tissue accumulation of the phe-tyr pathway metabolite homogentisic acid (HGA) is central to pathophysiology in the inherited disorder alkaptonuria (AKU). Accumulation of metabolites upstream of HGA, including tyrosine, occurs in patients on nitisinone, a licenced drug for AKU and hereditary tyrosinaemia type 1, which inhibits the enzyme responsible for HGA production. The aim of this study was to investigate the phe-tyr metabolite content of key biofluids and tissues in AKU mice on and off nitisinone to gain new insights into the biodistribution of metabolites in these altered metabolic states. The data show for the first time that HGA is present in bile in AKU (mean [±SD] = 1003[±410] μmol/L; nitisinone-treated AKU mean [±SD] = 45[±23] μmol/L). Biliary tyrosine, 3(4-hydroxyphenyl)pyruvic acid (HPPA) and 3(4-hydroxyphenyl)lactic acid (HPLA) are also increased on nitisinone. Urine was confirmed as the dominant elimination route of HGA in untreated AKU, but with indication of biliary excretion. These data provide new insights into pathways of phe-tyr metabolite biodistribution and metabolism, showing for the first time that hepatobiliary excretion contributes to the total pool of metabolites in this pathway. Our data suggest that biliary elimination of organic acids and other metabolites may play an underappreciated role in disorders of metabolism. We propose that our finding of approximately 3.8 times greater urinary HGA excretion in AKU mice compared with patients is one reason for the lack of extensive tissue ochronosis in the AKU mouse model.

CCN1 deficient stroma vasculature microct

CCN1-deficient stroma show disorganized collagen fibers. Digital pathology phenotypic quantification (PharmaNest, Inc) phenotypic fibrosis histologic heat chart (A) where each row represents a principal qFT, at the three phenotypic levels (collagen deposition, fibers morphometry, and fibrosis architecture). B, The Ph-FCS recapitulates all the qFTs for one sample, and quantifies the phenotype of fibrosis and its differences between CCN1-deficient (KO; n = 5, mean = 1.5, Std dev = 0.5) and wild-type (WT) mice (n = 6, mean = 4.6, Std dev = 0.9) groups (3.1 fold change, P < 0.001, Student t test). C, The collagen fiber morphometric scores recapitulates the differences at the morphometric level between KO (n = 5, mean = 1.6, Std dev = 0.5) and WT (n = 6, mean = 4.5, Std dev = 0.9) groups (2.9 fold change, P < 0.001, Student t test). D, Representative images with augmented digital pathology layers. “Entropy Texture” is one of the architectural phenotypes of fibrosis (39). KO, knockout.

Purpose (the aim of the study): This study addresses the critical question of the involvement of the cell surface endocytic receptor, the low-density lipoprotein receptor-related protein 1 (LRP1) in osteoarthritis (OA) in vivo. Our previous studies using cartilage explants from human patients have shown that LRP1 plays a vital role in maintaining healthy cartilage by removing various biologically active molecules from the tissue and degrading them in the cell. However, in OA cartilage, the function of the LRP1 receptor is largely lost due to increased proteolytic shedding of LRP1 ectodomain. Inhibition of proteolytic enzymes responsible for LRP1 shedding restores endocytic capacity of the cells and protect cartilage degradation ex vivo, but the role of LRP1 in OA development in vivo remains incompletely understood.