Search Results (35)

Background: The increasing prevalence of processed foods has significantly elevated dietary phosphorus intake globally, posing a risk to skeletal health. Elevated serum phosphate promotes parathyroid hormone (PTH) release, leading to bone resorption and decreased bone formation. Objective: This study investigated the influence of chronically elevated phosphorus intake on bone structure in rats with normal renal function, focusing on the Receptor Activator of Nuclear factor Kappa-B (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) pathway and its related components, leucine rich repeat containing G protein-coupled receptor 4 (LGR4), and R-spondins (RSPOs). Methods: Rats were fed a high-phosphorus diet, followed by assessment of the bone microstructure and of the expression of key signalling molecules. Results: Elevated phosphorus intake induced significant bone deterioration, particularly in the trabecular bone compartment, associated with alterations in the RANK/RANKL/OPG pathway and in the LGR4 and RSPO1 and RSPO4 signalling components in bone. Moreover, we also observed changes in RANKL, RSPO1 and RSPO4 serum levels in the rats that had received a high-phosphorus diet. Conclusions: These findings highlight the detrimental impact of excessive dietary phosphorus on skeletal health, even without renal impairment, and suggest that components of this pathway, particularly RSPO1 and RSPO4, could serve as potential biomarkers of bone deterioration. The widespread consumption of phosphorus-rich processed foods underscores the importance of nutritional education to mitigate these skeletal risks in industrialized populations. Full article

The Rotimer (rotifer-specific biopolymer) like SCO-spondin (R-SSPO/1), predicted as the main component of this biopolymer, is an adequate base for the design of functional small peptides. This macromolecule is interactive and protective against neurotoxic human-type beta-amyloid 1-42 aggregates (agg-Aβ). The current work presents biological investigations and predictable molecular interaction analysis of DSSNDL and PNCRDGSDE peptides that were synthesized based on the sequences of R-SSPO/1. Viability assays (NADH-dependent cellular reduction capacity, intracellular esterase activity, and motility) were performed on differentiated neuro-type cell cultures (SH-SY5Y and PC12) and on Rotimer-depleted rotifers (Euchlanis dilatata and Lecane bulla). A control peptide (STTRPTGTT), not found in Rotimer, was also included in the study. All three peptides are present in both rotifer and human proteomes. Among these small molecules, DSSNDL showed a significant protective effect against the toxicity of agg-Aβ both in vitro and in vivo and presumably interacted with its aggregates. The stagogram analysis of amyloid–peptide complexes and the possible bonding competition of these small molecules against aggregation-specific dyes on agg-Aβ surface suggest that DSSNDL affects the properties of these neurotoxic macromolecules. This effective hexapeptide can serve as a promising candidate for further investigations into the inactivation of beta-amyloid toxicity. Full article

The regulator of the canonical Wnt pathway, leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), is expressed in the stem cell compartment of several tissues and overexpressed in different human carcinomas. The isoform of the stem cell marker LGR5, named LGR5Δ5 and first described by our group, is associated with prognosis and metastasis in oral squamous cell carcinoma (OSCC) and soft tissue sarcoma (STS). In a proof-of-principle analysis, the function of LGR5Δ5 was investigated in HEK293T cells, a model cell line of the Wnt pathway, compared to full-length LGR5 (FL) expression. The CRISPR/CAS knockout of LGR5 and LGR4 (thereby avoiding the side effects of LGR4) resulted in a loss of Wnt activity that cannot be restored by LGR5Δ5 but by LGR5FL rescue. The ability to migrate was not affected by LGR5Δ5, but was reduced by LGR5FL overexpression. The CRISPR/CAS of LGR4 and 5 induced radiosensitization, which was enhanced by the overexpression of LGR5FL or LGR5Δ5. RNA sequencing analysis revealed a significant increase in the ligand R-spondin 1 (RSPO1) level by LGR5Δ5. Furthermore, LGR5Δ5 appears to be involved in the regulation of genes related to the cytoskeleton, extracellular matrix stiffness, and angiogenesis, while LGR5FL is associated with the regulation of collagens and histone proteins. Full article

Background/Objectives: Wnt signaling pathways are essential in various biological processes, including embryonic development and tissue homeostasis, and are implicated in many diseases. The R-Spondin (RSpo) family, particularly RSpo1, plays a significant role in modulating Wnt signaling. This study aims to explore how RSpo1 binding to astrocytic LGR6 receptors influences central nervous system (CNS) homeostasis, particularly in the context of inflammation. Methods: Human-induced pluripotent stem cell-derived astrocytes were treated with RSpo1 to assess its impact on inflammatory cytokine release. A proteomic analysis was conducted using a Human Cytokine Array Kit to measure differential protein expression. Pathway enrichment analysis was performed to identify affected signaling pathways. Results: RSpo1 treatment led to a suppression of inflammatory cytokines such as IL-10, IFN-γ, and IL-23 in astrocytes, while TNF-α and CXCL12 levels were increased. Pathway analysis revealed significant alterations in key signaling pathways, including cytokine–cytokine receptor interaction, chemokine signaling, and TNF signaling pathways, suggesting RSpo1’s role in modulating immune responses within the CNS. Conclusions: RSpo1 significantly influences inflammatory responses in astrocytes by modulating cytokine release and altering key signaling pathways. These findings enhance our understanding of the interaction between cell-specific Wnt signaling and CNS inflammation, suggesting potential therapeutic applications of RSpo1 in neuroinflammatory and neurodegenerative diseases. Full article

WNT3A is an intestinal ligand triggering the Wnt/β-catenin (Wnt) pathway, which can be enhanced by R-spondin 1 (RSPO1) through the RSPO1–LGR axis or antagonized by the adenomatous polyposis coli (APC) protein supporting β-catenin-degradation. Wnt interplays with several pathways including PI3K/mTOR (mTOR). In this study, we evaluated the influence of WNT3A-commercial and home-made culture media and RSPO1 protein on the Wnt and mTOR interplay in non-APC and APC-mutated intestinal patient-derived organoids (PDOs). Normal mucosa (NM) of sporadic CRC and FAP PDOs were cultured with: WNT3A-lacking/containing commercial (A/A+B) or home-made (BASAL/WNT3A-conditioned medium (CM)±RSPO1) media. In non-APC-mutated-PDOs (CRC-NM), WNT3A-CM, over commercial A+B, strongly activated Wnt-target-genes CCND1 and c-MYC. Most importantly, the addition of RSPO1 to home-made WNT3A-CM or A+B led to the downregulation of the mTOR-downstream-effector phospho-S6 ribosomal protein (p-S6R), highlighting the activation of the RSPO1–pS6R in both non-APC (CRC-NM) and APC-mutated (FAP-NM) PDOs, independently from LGR5 gene expression modulation. Our work demonstrates that home-made WNT3A-CM strongly impacts the crosstalk between Wnt and mTOR over commercial media, and proposes RSPO1 as a key regulator of the RSPO1–p-S6R axis in both non-APC and APC-mutated PDOs. Together, these findings represent an important methodological guide for scientists working in these fields to select the most appropriate intestinal PDO media. Full article

Impairment of the intestinal epithelial barrier is frequently seen as collateral damage in various local and systemic inflammatory conditions. The inflammatory process is characterized by reciprocal interactions between the host intestinal epithelium and mucosal innate immune cells, e.g., macrophages. This article provides step-by-step instructions on how to set up a murine enteroid–macrophage co-culture by culturing cellular elements in proximity separated by a porous membrane. Unlike previously published co-culture systems, we have combined enteroids grown from C57BL6j mice with syngeneic bone marrow-derived macrophages to preclude potential allo-reactions between immune cells and epithelium. Transformation of intestinal crypts into proliferative enteroids was achieved by cultivation in Wnt3a-Noggin-R-Spondin-conditioned medium supplemented with ROCK inhibitor Y-27632. The differentiated phenotype was promoted by the use of the Wnt3-deprived EGF-Noggin-R-Spondin medium. The resulting co-culture of primary cells can be employed as a basic model to better understand the reciprocal relationship between intestinal epithelium and macrophages. It can be used for in vitro modelling of mucosal inflammation, mimicked by stimulation of macrophages either while being in co-culture or before being introduced into co-culture, to simulate enterogenic sepsis or systemic conditions affecting the intestinal tract. Full article

Crohn’s disease is a chronic, debilitating, inflammatory bowel disease. Here, we report a critical role of phospholipase C-β3 (PLC-β3) in intestinal homeostasis. In PLC-β3-deficient mice, exposure to oral dextran sodium sulfate induced lethality and severe inflammation in the small intestine. The lethality was due to PLC-β3 deficiency in multiple non-hematopoietic cell types. PLC-β3 deficiency resulted in reduced Wnt/β-catenin signaling, which is essential for homeostasis and the regeneration of the intestinal epithelium. PLC-β3 regulated the Wnt/β-catenin pathway in small intestinal epithelial cells (IECs) at transcriptional, epigenetic, and, potentially, protein–protein interaction levels. PLC-β3-deficient IECs were unable to respond to stimulation by R-spondin 1, an enhancer of Wnt/β-catenin signaling. Reduced expression of PLC-β3 and its signature genes was found in biopsies of patients with ileal Crohn’s disease. PLC-β regulation of Wnt signaling was evolutionally conserved in Drosophila. Our data indicate that a reduction in PLC-β3-mediated Wnt/β-catenin signaling contributes to the pathogenesis of ileal Crohn’s disease. Full article

Fibroblast growth factor 5 (FGF5) plays key roles in promoting the transition from the anagen to catagen during the hair follicle cycle. The sheep serves as an excellent model for studying hair growth and is frequently utilized in various research processes related to human skin diseases. We used the CRISPR/Cas9 system to generate four FGF5-edited Dorper sheep and only low levels of FGF5 were detected in the edited sheep. The density of fine wool in GE sheep was markedly increased, and the proportion of fine wool with a diameter of 14.4–20.0 μm was significantly higher. The proliferation signal in the skin of gene-edited (GE) sheep was stronger than in wild-type (WT) sheep. FGF5 editing decreased cortisol concentration in the skin, further activated the activity of antioxidant enzymes such as Glutathione peroxidase (GSH-Px), and regulated the expression of Wnt signaling pathways containing Wnt agonists (Rspondins, Rspos) and antagonists (Notum) in hair regeneration. We suggest that FGF5 not only mediates the activation of antioxidant pathways by cortisol, which constitutes a highly coordinated microenvironment in hair follicle cells, but also influences key signals of the Wnt pathway to regulate secondary hair follicle (SHF) development. Overall, our findings here demonstrate that FGF5 plays a significant role in regulating SHF growth in sheep and potentially serves as a molecular marker of fine wool growth in sheep breeding. Full article

Vascular calcification has a global health impact that is closely linked to bone loss. The Receptor Activator of Nuclear Factor Kappa B (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) system, fundamental for bone metabolism, also plays an important role in vascular calcification. The Leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4), a novel receptor for RANKL, regulates bone remodeling, and it appears to be involved in vascular calcification. Besides RANKL, LGR4 interacts with R-spondins (RSPOs), which are known for their roles in bone but are less understood in vascular calcification. Studies were conducted in rats with chronic renal failure fed normal or high phosphorus diets for 18 weeks, with and without control of circulating parathormone (PTH) levels, resulting in different degrees of aortic calcification. Additionally, vascular smooth muscle cells (VSMCs) were cultured under non-calcifying (1 mM phosphate) and calcifying (3 mM phosphate) media with different concentrations of PTH. To explore the role of RANKL in VSMC calcification, increasing concentrations of soluble RANKL were added to non-calcifying and calcifying media. The effects mediated by RANKL binding to its receptor LGR4 were investigated by silencing the LGR4 receptor in VSMCs. Furthermore, the gene expression of the RANK/RANKL/OPG system and the ligands of LGR4 was assessed in human epigastric arteries obtained from kidney transplant recipients with calcification scores (Kauppila Index). Increased aortic calcium in rats coincided with elevated systolic blood pressure, upregulated Lgr4 and Rankl gene expression, downregulated Opg gene expression, and higher serum RANKL/OPG ratio without changes in Rspos gene expression. Elevated phosphate in vitro increased calcium content and expression of Rankl and Lgr4 while reducing Opg. Elevated PTH in the presence of high phosphate exacerbated the increase in calcium content. No changes in Rspos were observed under the conditions employed. The addition of soluble RANKL to VSMCs induced genotypic differentiation and calcification, partly prevented by LGR4 silencing. In the epigastric arteries of individuals presenting vascular calcification, the gene expression of RANKL was higher. While RSPOs show minimal impact on VSMC calcification, RANKL, interacting with LGR4, drives osteogenic differentiation in VSMCs, unveiling a novel mechanism beyond RANKL-RANK binding. Full article

WNT/β-catenin signaling is essential for colon cancer development and progression. WNT5A (ligand of non-canonical WNT signaling) and its mimicking peptide Foxy5 impair β-catenin signaling in colon cancer cells via unknown mechanisms. Therefore, we investigated whether and how WNT5A signaling affects two promoters of β-catenin signaling: the LGR5 receptor and its ligand RSPO3, as well as β-catenin activity and its target gene VEGFA. Protein and gene expression in colon cancer cohorts were analyzed by immunohistochemistry and qRT-PCR, respectively. Three colon cancer cell lines were used for in vitro and one cell line for in vivo experiments and results were analyzed by Western blotting, RT-PCR, clonogenic and sphere formation assays, immunofluorescence, and immunohistochemistry. Expression of WNT5A (a tumor suppressor) negatively correlated with that of LGR5/RSPO3 (tumor promoters) in colon cancer cohorts. Experimentally, WNT5A signaling suppressed β-catenin activity, LGR5, RSPO3, and VEGFA expression, and colony and spheroid formations. Since β-catenin signaling promotes colon cancer stemness, we explored how WNT5A expression is related to that of the cancer stem cell marker DCLK1. DCLK1 expression was negatively correlated with WNT5A expression in colon cancer cohorts and was experimentally reduced by WNT5A signaling. Thus, WNT5A and Foxy5 decrease LGR5/RSPO3 expression and β-catenin activity. This inhibits stemness and VEGFA expression, suggesting novel treatment strategies for the drug candidate Foxy5 in the handling of colon cancer patients. Full article

Matricellular proteins are secreted extracellular proteins that bear no primary structural functions but play crucial roles in tissue remodeling during development, homeostasis, and aging. Despite their low expression after birth, matricellular proteins within skin compartments support the structural function of many extracellular matrix proteins, such as collagens. In this review, we summarize the function of matricellular proteins in skin stem cell niches that influence stem cells’ fate and self-renewal ability. In the epidermal stem cell niche, fibulin 7 promotes epidermal stem cells’ heterogeneity and fitness into old age, and the transforming growth factor-β—induced protein ig-h3 (TGFBI)—enhances epidermal stem cell growth and wound healing. In the hair follicle stem cell niche, matricellular proteins such as periostin, tenascin C, SPARC, fibulin 1, CCN2, and R-Spondin 2 and 3 modulate stem cell activity during the hair cycle and may stabilize arrector pili muscle attachment to the hair follicle during piloerections (goosebumps). In skin wound healing, matricellular proteins are upregulated, and their functions have been examined in various gain-and-loss-of-function studies. However, much remains unknown concerning whether these proteins modulate skin stem cell behavior, plasticity, or cell–cell communications during wound healing and aging, leaving a new avenue for future studies. Full article

Ampullary adenocarcinoma is a rare malignancy that lacks standard systemic treatment. We describe a case of recurrent metastatic ampullary adenocarcinoma of the pancreaticobiliary subtype treated with nanoparticle albumin-bound (nab)-paclitaxel and gemcitabine as first-line treatment. This report also highlights the molecular profile of the ampullary adenocarcinoma and circulating tumor DNA (ctDNA). This is a case of pancreaticobiliary ampullary adenocarcinoma in a 67-year-old woman who initially presented with painless jaundice. Endoscopic and imaging evaluation revealed biliary ductal dilation secondary to an ampullary mass. Pathology confirmed the diagnosis of ampullary adenocarcinoma of the pancreaticobiliary subtype. She underwent surgical resection of the tumor, followed by adjuvant chemotherapy with gemcitabine and capecitabine. The tumor subsequently recurred in the liver. She received palliative chemotherapy with nab-paclitaxel and gemcitabine, resulting in an objective tumor response for 14 months. Molecular profiling of the tumor and ctDNA revealed a novel MATN2-RSPO RNA fusion and a novel NTRK3 mutation, respectively. Our report suggests that long-term durable response can be achieved in metastatic pancreaticobiliary ampullary adenocarcinoma using nab-paclitaxel and gemcitabine. Molecular profiling of the tumor identified a novel R-Spondin2 RNA fusion and NTRK3 mutation that can be potentially targeted for treatment. Full article

The Chinese soft-shelled turtle, Pelodiscus sinensis, is an important aquaculture species in China that exhibits distinct sexual dimorphism; male individuals are economically more valuable than females. In vertebrates, several R-spondin family proteins have been associated with sex differentiation mechanisms; however, their involvement in P. sinensis sex differentiation is unclear. Exogenous hormones such as estradiol (E2) also influence the sex differentiation of P. sinensis and induce sexual reversal. In the present study, we investigated the effects of E2 on the embryonic development of P. sinensis and the expression of R-spondin 2 (Rspo2) and R-spondin 3 (Rspo3). We amplified P. sinensis Rspo2 and Rspo3 and analyzed their expression patterns in different tissues. Comparative analyses with protein sequences from other species elucidated that P. sinensis RSPO2 and RSPO3 sequences were conserved. Moreover, phylogenetic analysis revealed that P. sinensis RSPO2 and RSPO3 were closely related to these two proteins from other turtle species. Furthermore, Rspo2 and Rspo3 were highly expressed in the brain and gonads of adult turtles, with significantly higher expression in the ovaries than in the testes (p < 0.05). We also evaluated the expression of Rspo2 and Rspo3 after the administration of three concentrations of E2 (1.0, 5.0, and 10.0 mg/mL) to turtle eggs during embryonic development. The results revealed that E2 upregulated Rspo2 and Rspo3, and the expression trends varied during different embryonic developmental stages (stages 13–20). These findings lay the groundwork for future investigations into the molecular mechanisms involved in the sex differentiation of Chinese soft-shelled turtles. Full article

The mechanism of sex determination in chickens, especially the molecular mechanism of female ovarian development, has not yet been fully elucidated. Previous studies have shown that RSPO1, which is associated with ovarian development in mammals, might have a conserved role in chickens. In this study, we systematically investigated the spatiotemporal expression pattern of RSPO1 in various tissues, especially gonads, of male and female chicken embryos using qPCR and Western blotting, and we explored its correlation with the expression of key genes in the estrogen pathway using drug treatment or gene overexpression in vivo and in vitro. Our results reveal that RSPO1 was widely expressed in all examined tissues of chicken embryos, showing a female bias in gonadal tissues at both the mRNA and protein levels. Surprisingly, RSPO1 was not differentially expressed between male and female gonadal cells with fadrozole-induced estrogen pathway blockades, and furthermore, estradiol-induced estrogen stimulation altered the expression of RSPO1. In addition, overexpression of RSPO1 in gonadal cells induced the mRNA expression of its downstream target genes, Wnt family member 4 (WNT4) and Catenin beta 1 (CTNNB1), and that of estrogen receptor α (ERα), an estrogen pathway gene. In summary, this study provided new evidence for elucidating the role of RSPO1 in ovarian development in poultry. Full article

All-trans retinoic acid (ATRA) promotes myoblast differentiation into myotubes. Leucine-rich repeat-containing G-protein-coupled receptor 6 (LGR6) is a candidate ATRA-responsive gene; however, its role in skeletal muscles remains unclear. Here, we demonstrated that during the differentiation of murine C2C12 myoblasts into myotubes, Lgr6 mRNA expression transiently increased before the increase in the expression of the mRNAs encoding myogenic regulatory factors, such as myogenin, myomaker, and myomerger. The loss of LGR6 decreased the differentiation and fusion indices. The exogenous expression of LGR6 up to 3 and 24 h after the induction of differentiation increased and decreased the mRNA levels of myogenin, myomaker, and myomerger, respectively. Lgr6 mRNA was transiently expressed after myogenic differentiation in the presence of a retinoic acid receptor α (RARα) agonist and an RARγ agonist in addition to ATRA, but not in the absence of ATRA. Furthermore, a proteasome inhibitor or Znfr3 knockdown increased exogenous LGR6 expression. The loss of LGR6 attenuated the Wnt/β-catenin signaling activity induced by Wnt3a alone or in combination with Wnt3a and R-spondin 2. These results indicate that LGR6 promotes myogenic differentiation and that ATRA is required for the transient expression of LGR6 during differentiation. Furthermore, LGR6 expression appeared to be downregulated by the ubiquitin–proteasome system involving ZNRF3. Full article