Biology subjects

Riobo-Del Galdo, N. A.

Publications and source records attributed to Riobo-Del Galdo, N. A..

6 recordsLinked to original sources

bioRxiv2026

Aurora A kinase activation contributes to the fibrotic phenotype in Systemic Sclerosis through primary cilia shortening

BackgroundSystemic sclerosis (SSc) is a severe autoimmune disease characterised by progressive fibrosis driven by fibroblast activation. Primary cilia, key hubs for profibrotic signalling, are markedly shortened in SSc fibroblasts, but the mechanisms underlying this phenotype remain unclear. This study aimed to define the signalling pathways responsible for primary cilia shortening and fibroblast activation in SSc. MethodsPrimary dermal fibroblasts from SSc patients and healthy controls were analysed for cilia incidence and length by immunofluorescence, profibrotic marker expression by qPCR, and contractility using gel contraction assays. Cells were treated with TGF{beta}1 and pharmacological inhibitors targeting AURKA, HDAC6, ROCK2, and Smad3 signalling. CAV1-silenced fibroblasts were used as an in vitro model of SSc. ResultsMaintenance of the constitutively short primary cilia phenotype in SSc fibroblasts did not require active TGF{beta} signalling. However, TGF{beta}1 induced reversible cilia shortening in healthy fibroblasts and further shortened cilia in SSc fibroblasts to a similar final length, mediated by Rho/ROCK2 rather than canonical Smad3-dependent signalling. Constitutive cilia shortening in SSc was driven by aberrant AURKA activity upstream of HDAC6, promoting ciliary disassembly. Pharmacological inhibition of AURKA or HDAC6 selectively elongated cilia in SSc fibroblasts, reduced profibrotic marker expression, and abrogated fibroblast contractility. CAV1-silenced fibroblasts similarly exhibited constitutive cilia shortening that was reversed by AURKA inhibition without affecting healthy cells. ConclusionsAberrant activation of the AURKA/HDAC6 axis maintains short primary cilia and promotes fibroblast activation in SSc. These findings reveal a mechanistic link between cilia morphology and fibrosis and identify AURKA as a potential therapeutic target for SSc-associated tissue remodelling.

cell biology↗

bioRxiv2026

Imbalance of Ciliary Programs Drives Fibroblast Differentiation and Fibrotic Signaling in Systemic Sclerosis

Systemic sclerosis (SSc) is a chronic fibrotic disease characterized by accumulation of activated profibrotic myofibroblasts in multiple organs. The mechanisms triggering pathogenic fibroblast to myofibroblast reprogramming in SSc, and maintaining the activated myofibroblast state, are not well known. Recent studies show that primary cilia (PC), solitary sensory organelles that integrate diverse chemical and mechanical signaling pathways, may regulate fibroblast fates. Here, using an orthogonal multimodal strategy spanning human cohorts, single-cell trajectories, and targeted perturbation models, we identify a fundamental imbalance in cilia-program dynamics as a unifying driver of fibrotic activation in SSc. Meta-analysis of skin biopsies integrating eight microarray datasets and two independent scRNA-seq cohorts, revealed a conserved 15-gene cilia signature that is altered in SSc. Single-cell trajectory mapping resolved a principal progenitor[->] secretory-like [->] myofibroblast differentiation axis, where SSc fibroblasts displayed spatially dysregulated ciliary programs that established a prolonged, disassembly-dominant "cilia-off" state. Notably, this ciliary imbalance appeared to emerge before the transition to fibrotic gene programs, positioning cilia disruption as an initiating, not secondary, event in fibroblast programming. Consistent with these transcriptomic findings, PC length was significantly reduced in SSc skin and cultured fibroblasts. Mechanistically, genetic and pharmacological perturbation studies demonstrated that cilia disruption is sufficient to drive fibrotic programs. These data establish a reciprocal regulatory framework in which shortened cilia promote sustained activation of TGF-{beta}-Hippo feed-forward signaling, driving fibroblast-to-myofibroblast transition and amplifying fibrotic responses. Together, these findings position the imbalance between ciliary assembly and disassembly as a central determinant of fibrotic fibroblast fate in SSc. Moreover, they indicate that therapeutically stabilizing primary cilia, "ciliotherapy", can reset pathological fibroblast trajectories and represents a promising antifibrotic strategy for SSc and other fibrotic conditions. TeaserDisrupted Primary Cilia Drives Pathophysiology of Systemic Sclerosis.

cell biology↗

bioRxiv2023

The Hedgehog receptors PTCH1 and PTCH2 exist as active homomeric and heteromeric complexes

With the importance of Hedgehog signalling in embryonic development, tissue homeostasis and disease, understanding the molecular mechanisms of signal transduction is paramount for the design of specific, effective therapeutics. The Hedgehog receptor PTCH1 and the less studied PTCH2 isoform are evolutionarily related to bacterial RND permeases and sterol sensing proteins, which mobilise hydrophobic compounds powered by a cation gradient. Here we demonstrate that, in the active state, PTCH1 and PTCH2 form homomeric and heteromeric complexes that are inhibited by binding of Sonic Hedgehog. We show that PTCH2, unlike PTCH1, appears to have minimal cholesterol transport activity, but that conserved residues involved in cation transport are essential for its function. Heteromeric PTCH1-PTCH2 complexes depend on PTCH1s cholesterol transport capacity, but the cation transport can be provided in trans by PTCH2, suggesting that some deleterious mutations in either isoform can be silenced by formation of heteromers, enhancing the robustness of this signal transduction system. These findings provide the molecular basis for the intriguing behaviour of PTCH2 as semi-redundant and partially overlapping in function with PTCH1 and explain the dominant negative effect of mutations that disrupt the PTCH2 cation transport triad in rare cases of cancer.

biochemistry↗

bioRxiv2023

Towards Modular Engineering of Cell Signalling: Topographically-Textured Microparticles Induce Osteogenesis via Activation of Canonical Hedgehog Signalling

Polymer microparticles possess great potential as functional building blocks for advanced bottom-up engineering of complex tissues. Tailoring the three-dimensional architectural features of culture substrates has been shown to induce osteogenesis in mesenchymal stem cells in vitro, but the molecular mechanisms underpinning this remain unclear. This study proposes a mechanism linking the activation of Hedgehog signalling to the osteoinductive effect of surface-engineered, topographically-textured polymeric microparticles. In this study, mesenchymal progenitor C3H10T1/2 cells were cultured on smooth and dimpled poly(D,L-lactide) microparticles to assess differences in viability, cellular morphology, proliferation, and expression of a range of Hedgehog signalling components and osteogenesis-related genes. Dimpled microparticles induced osteogenesis and activated the Hedgehog signalling pathway relative to smooth microparticles and 2D-cultured controls without the addition of exogenous biochemical factors. We observed upregulation of the osteogenesis markers Runt-related transcription factor2 (Runx2) and bone gamma-carboxyglutamate protein 2 (Bglap2), as well as the Hedgehog signalling components, glioma associated oncogene homolog 1 (Gli1), Patched1 (Ptch1), and Smoothened (Smo). Treatment with the Smo antagonist KAAD-cyclopamine confirmed the involvement of Smo in Gli1 target gene activation, with a significant reduction in the expression of Gli1, Runx2 and Bglap2 (p[≤]0.001) following KAAD-cyclopamine treatment. Overall, our study demonstrates the role of the topographical microenvironment in the modulation of Hedgehog signalling, highlighting the potential for tailoring substrate topographical design to offer cell-instructive 3D microenvironments. Topographically-textured microparticles allow the modulation of Hedgehog signalling in vitro without adding exogenous biochemical agonists, thereby eliminating potential confounding artefacts in high-throughput drug screening applications. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=134 HEIGHT=200 SRC="FIGDIR/small/549481v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@cdeb88org.highwire.dtl.DTLVardef@1224098org.highwire.dtl.DTLVardef@1bee1b1org.highwire.dtl.DTLVardef@d34708_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2021

Chloride intracellular channel 4 (CLIC4) expression is transcriptionally regulated by crosstalk of the TGF-beta, Wnt and Hedgehog signalling pathways

Chloride intracellular channel 4 (CLIC4) is a recently discovered driver of fibroblast activation in Scleroderma (SSc) and cancer-associated fibroblasts. CLIC4 expression and activity are regulated by TGF-{beta} signalling through the SMAD3 transcription factor. In view of the aberrant activation of canonical Wnt and Hedgehog (Hh) signalling in fibrosis, we investigated their role in CLIC4 upregulation. Here, we show Wnt3a/{beta}-catenin and Smoothened/GLI signalling cooperate with SMAD3 to regulate CLIC4 expression in normal dermal fibroblasts, and that inhibition of SMAD3 expression or activity abolishes Wnt and Hh-dependent CLIC4 induction. We further show that expression of the profibrotic marker -smooth muscle actin strongly correlates with CLIC4 expression in dermal fibroblasts. Our data highlight novel mechanisms that regulate CLIC4 expression that present targetable pathways to prevent fibroblast activation in SSc and other fibrotic conditions.

cell biology↗

bioRxiv2020

Ubiquitin-protein ligase Ubr5 cooperates with Hedgehog signalling to promote skeletal tissue homeostasis

Mammalian Hedgehog (HH) signalling pathway plays an essential role in tissue homeostasis and its deregulation is linked to rheumatological disorders. UBR5 is the mammalian homologue of the E3 ubiquitin-protein ligase Hyd, a negative regulator of the Hh-pathway in Drosophila. To investigate a possible role of UBR5 in regulation of the musculoskeletal system through modulation of mammalian HH signaling, we created a mouse model for specific loss of Ubr5 function in limb bud mesenchyme. Our findings revealed a role for UBR5 in maintaining cartilage homeostasis and suppressing metaplasia. Ubr5 loss of function resulted in progressive and dramatic articular cartilage degradation, enlarged, abnormally shaped sesamoid bones and extensive heterotopic tissue metaplasia linked to calcification of tendons and ossification of synovium. Genetic suppression of smoothened (Smo), a key mediator of HH signalling, dramatically enhanced the Ubr5 mutant phenotype. Analysis of HH signalling in both mouse and cell model systems revealed that loss of Ubr5 stimulated canonical HH-signalling while also increasing PKA activity. In addition, human osteoarthritic samples revealed similar correlations between UBR5 expression, canonical HH signalling and PKA activity markers. Our studies identified a crucial function for the Ubr5 gene in the maintenance of skeletal tissue homeostasis and an unexpected mode of regulation of the HH signalling pathway. Author SummaryUbiquitin ligases modify proteins post-translationally which is essential for a variety of cellular processes. UBR5 is an E3 ubiquitin ligase and in Drosophila is a regulator of Hedgehog signaling. In mammals, the Hedgehog (HH) signalling pathway, among many other roles, plays an essential role in tissue maintenance, a process called homeostasis. A murine genetic system was developed to specifically eliminate UBR5 function from embryonic limb tissue that subsequently forms bone and connective tissue (ligaments and tendons). This approach revealed that UBR5 operates as a potent suppressor of excessive growth of normal cartilage and bone and prevents formation of bone in ectopic sites in connective tissue near the knees and ankle joints. In contrast to abnormal growth, UBR5 inhibits degradation of the articular cartilage that cushions the knee joint leading to extensive exposure of underlying bone. Furthermore, Ubr5 interacts with smoothened, a component of the HH pathway, identifying UBR5 as a regulator of mammalian HH signaling in the postnatal musculoskeletal system. In summary, this work shows that UBR5 interacts with the HH pathway to regulate skeletal homeostasis in and around joints of the legs and identifies targets that may be harnessed for biomedical engineering and clinical applications.

genetics↗