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Background: Limb-girdle muscular dystrophy R1 calpain 3-related (LGMDR1) is an autosomal recessive muscular dystrophy due to mutations in the CAPN3 gene. While the pathophysiology of this disease has not been clearly established yet, Wnt and mTOR signaling pathways impairment in LGMDR1 muscles has been reported., Results: A reduction in Akt phosphorylation ratio and upregulated expression of proteins implicated in glycolysis (HK-II) and in fructose and lactate transport (GLUT5 and MCT1) in LGMDR1 muscle was observed. In vitro analysis to establish mitochondrial and glycolytic functions of primary cultures were performed, however, no differences between control and patients were observed. Additionally, gene expression analysis showed a lack of correlation between primary myoblasts/myotubes and LGMDR1 muscle while skin fibroblasts and CD56- cells showed a slightly better correlation with muscle. FRZB gene was upregulated in all the analyzed cell types (except in myoblasts)., Conclusions: Proteins implicated in metabolism are deregulated in LGMDR1 patients' muscle. Obtained results evidence the limited usefulness of primary myoblasts/myotubes for LGMDR1 gene expression and metabolic studies. However, since FRZB is the only gene that showed upregulation in all the analyzed cell types it is suggested its role as a key regulator of the pathophysiology of the LGMDR1 muscle fiber. The Wnt signaling pathway inactivation, secondary to FRZB upregulation, and GLUT5 overexpression may participate in the impaired adipogenesis in LGMD1R patients., (© 2023. Institut National de la Santé et de la Recherche Médicale (INSERM).)

Background & Aims: Cholangiocarcinoma (CCA) is a biliary malignancy linked to genetic and epigenetic abnormalities, such as hypermethylation of SOX17 promoter. Here, the role of SOX17 in cholangiocyte differentiation and cholangiocarcinogenesis was studied., Methods: SOX17 expression/function was evaluated along the differentiation of human induced pluripotent stem cells (iPSC) into cholangiocytes, in the dedifferentiation process of normal human cholangiocytes (NHC) in culture and in cholangiocarcinogenesis. Lentiviruses for SOX17 overexpression or knockdown were used. Gene expression and DNA methylation profiling were performed., Results: SOX17 expression is induced in the last stage of cholangiocyte differentiation from iPSC and regulates the acquisition of biliary markers. SOX17 becomes downregulated in NHC undergoing dedifferentiation; experimental SOX17 knockdown in differentiated NHC downregulated biliary markers and promoted baseline and Wnt-dependent proliferation. SOX17 expression is lower in human CCA than in healthy tissue, which correlates with worse survival after tumor resection. In CCA cells, SOX17 overexpression decreased their tumorigenic capacity in murine xenograft models, which was related to increased oxidative stress and apoptosis. In contrast, SOX17 overexpression in NHC did not affect their survival but inhibited their baseline proliferation. In CCA cells, SOX17 inhibited migration, anchorage-independent growth and Wnt/β-catenin-dependent proliferation, and restored the expression of biliary markers and primary cilium length. In human CCA, SOX17 promoter was found hypermethylated and its expression inversely correlates with the methylation grade. In NHC, Wnt3a decreased SOX17 expression in a DNMT-dependent manner, whereas in CCA, DNMT1 inhibition or silencing upregulated SOX17., Conclusions: SOX17 regulates the differentiation and maintenance of the biliary phenotype and functions as a tumor suppressor for CCA, being a potential prognostic marker and a promising therapeutic target., Lay Summary: Understanding the molecular mechanisms involved in the pathogenesis of CCA is key in finding new valuable diagnostic and prognostic biomarkers, as well as therapeutic targets. This study provides evidence that SOX17 regulates the differentiation and maintenance of the biliary phenotype, and its downregulation promotes their tumorigenic transformation. SOX17 acts as a tumor suppressor in CCA and its genetic, molecular and/or pharmacological restoration may represent a new promising therapeutic strategy. Moreover, SOX17 expression correlates with the outcome of patients after tumor resection, being a potential prognostic biomarker., (Copyright © 2017 European Association for the Study of the Liver. All rights reserved.)

Limb-girdle muscular dystrophy type 2A (LGMD2A) is characterised by muscle wasting and progressive degeneration of proximal muscles because of mutations in the CAPN3 gene. However, the underlying pathophysiological mechanisms of muscle degeneration are still not well understood. The objective of this study was to assess the relevance of genes with differential expression in the muscle of LGMD2A patients. For this purpose, we analysed their in vitro expression in primary cultures of human myoblasts and myotubes. Abnormal fusion was observed in the myotubes of these patients, which may be explained by the lack of physiological replacement of integrin β1D. Owing to this observation, we focused on deregulated genes coding proteins that directly interact with integrin, ITGB1BP2 and CD9, as well as FRZB gene, because of its in vitro upregulation in myotubes. Silencing studies established that these genes are closely regulated, CD9 and FRZB being positive regulators of the expression of ITGB1BP2, and in turn, this gene being a negative regulator of the expression of FRZB. Interestingly, we observed that FRZB regulates integrin β1D expression, its silencing increasing integrin β1D expression to levels similar to those in controls. Finally, the administration of LiCl, an enhancer of the Wnt-signalling pathway showed similar experimentally beneficial effects, suggesting FRZB silencing or LiCl administration as potential therapeutic targets, though further studies are required.

Cholangiocarcinoma (CCA) is a heterogeneous group of dysplastic disorders affecting the biliary epithelium. It is the second most common primary liver tumor which accounts for around 3% of all gastrointestinal cancers. CCA is very deadly due to its aggressiveness, late diagnosis and high chemoresistance. The incidence is increasing worldwide and the therapeutic options are very limited. Radiotherapy, chemotherapy, surgery and/or liver transplantation may be indicated in patients who meet certain criteria, but chances of success are low. There is therefore increasing interest in understanding the molecular mechanisms involved in the pathogenesis of this cancer type and in identifying new targets for therapy. Current strategies are based on targeting key signaling pathways involved in proliferation, survival, apoptosis and migration. In this review, the most relevant molecular mechanisms involved in the pathogenesis of CCA are discussed and the main preclinical and clinical studies are highlighted. Moreover, future directions in basic and clinical research are indicated., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Objective: Polycystic liver diseases (PCLDs) are genetic disorders characterised by progressive bile duct dilatation and/or cyst development. Their pathogenesis is a consequence of hyperproliferation, hypersecretion and microRNA alterations in cholangiocytes. Here we evaluate the role of matrix metalloproteases (MMPs) in the hepatic cystogenesis of PCLDs., Design: Metalloprotease activity was measured by microfluorimetric assays in normal and polycystic cholangiocyte cultures from humans and rats, and gene expression by real time quantitative PCR. The role of cytokines, oestrogens and growth factors present in the cystic fluid of PCLD patients was evaluated for MMP activity. The MMP inhibitor marimastat was examined for cystic expansion in vitro and in polycystic kidney (PCK) rats., Results: Polycystic human and rat cholangiocytes displayed increased MMP activity, which was associated with increased mRNA levels of different MMPs. Interleukin (IL)-6 and IL-8, and 17β-oestradiol, all stimulated MMP activity in human cholangiocytes. The presence of antibodies against IL-6 and/or IL-8 receptor/s inhibited baseline MMP hyperactivity of polycystic human cholangiocytes but had no effect on normal human cholangiocytes. MMP-3 was overexpressed in cystic cholangiocytes from PCLD human and PCK rat livers by immunohistochemistry. Marimastat reduced MMP hyperactivity of polycystic human and rat cholangiocytes and blocked the cystic expansion of PCK cholangiocytes cultured in three-dimensions. Chronic treatment of 8-week-old PCK rats with marimastat inhibited hepatic cystogenesis and fibrosis., Conclusions: PCLDs are associated with cholangiocyte MMP hyperactivity resulting from autocrine/paracrine stimulation by IL-6 and IL-8. Inhibition of this MMP hyperactivity with marimastat decreased hepatic cystogenesis in vitro and in an animal model of PCLD, offering a potential therapeutic tool., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Limb-girdle muscular dystrophy type 2A (LGMD2A) due to mutations in the CAPN3 gene is one of the most common of autosomal recessive limb-girdle muscular dystrophies. We describe a patient who had a typical LGMD2A phenotype and posterior compartment involvement on MRI. Different genetic analyses were performed, including microarray analysis. There was an apparently homozygous mutation in exon 24, c.2465G>T, p.(*822Leuext62*), and a lack of correlation in the disease segregation analyses. This suggested the presence of a genomic rearrangement. In fact, a heterozygous deletion of the entire CAPN3 gene was found. This novel deletion comprised the terminal region of the GANC gene and the entire CAPN3 gene. This finding points out the need to reconsider and adapt our current strategy of molecular diagnosis in order to detect these types of genomic rearrangements that escape standard mutation screening procedures., (© 2014 Wiley Periodicals, Inc.)

Mutations in CAPN3 gene cause limb-girdle muscular dystrophy type 2A (LGMD2A) characterized by muscle wasting and progressive degeneration of scapular and pelvic musculature. Since CAPN3 knockout mice (C3KO) display features of muscle pathology similar to those features observed in the earliest-stage or preclinical LGMD2A patients, gene expression profiling analysis in C3KO mice was performed to gain insight into mechanisms of disease. Two different comparisons were carried out in order to determine, first, the differential gene expression between wild-type (WT) and C3KO soleus and, second, to identify the transcripts differentially expressed in aging muscles of WT and C3KO mice. The up/downregulation of two genes, important for normal muscle function, was identified in C3KO mice: the Ky gene, encoding a protease implicated in muscle development, and Park2 gene encoding an E3 ubiquitin ligase (parkin). The Ky gene was downregulated in C3KO muscles suggesting that Ky protease may play a complementary role in regulating muscle cytoskeleton homeostasis in response to changes in muscle activity. Park2 was upregulated in the aged WT muscles but not in C3KO muscles. Taking into account the known functions of parkin E3 ligase, it is possible that it plays a role in ubiquitination and degradation of atrophy-specific and damaged proteins that are necessary to avoid cellular toxicity and a cellular stress response in aging muscles.

Introduction: Limb-girdle muscular dystrophy type 2A (LGMD2A) is caused by a deficiency of calpain-3/p94. Although the symptoms in most LGMD2A patients are generally homogeneous, some variation in the severity and progression of the disease has been reported., Methods: We describe 2 patients who carry the same combination of compound heterozygous mutations (pG222R/pR748Q) and whose symptoms are exceptionally benign compared to homozygotes with each missense mutation., Results: The benign phenotype observed in association with the combined pG222R and pR748Q mutations suggested that it may result from a compensatory effect of compound heterozygosity rather than the individual mutations themselves. Our analyses revealed that these two mutations exert different effects on the protease activity of calpain-3, suggesting "molecular complementation" in these patients., Conclusion: We propose several hypotheses to explain how this specific combination of mutations may rescue the normal proteolytic activity of calpain-3, resulting in an exceptionally benign phenotype., (Copyright © 2011 Wiley Periodicals, Inc.)

Limb-girdle muscular dystrophy type 2A (LGMD2A) is a recessive genetic disorder caused by mutations in calpain 3 (CAPN3). Calpain 3 plays different roles in muscular cells, but little is known about its functions or in vivo substrates. The aim of this study was to identify the genes showing an altered expression in LGMD2A patients and the possible pathways they are implicated in. Ten muscle samples from LGMD2A patients with in which molecular diagnosis was ascertained were investigated using array technology to analyze gene expression profiling as compared to ten normal muscle samples. Upregulated genes were mostly those related to extracellular matrix (different collagens), cell adhesion (fibronectin), muscle development (myosins and melusin) and signal transduction. It is therefore suggested that different proteins located or participating in the costameric region are implicated in processes regulated by calpain 3 during skeletal muscle development. Genes participating in the ubiquitin proteasome degradation pathway were found to be deregulated in LGMD2A patients, suggesting that regulation of this pathway may be under the control of calpain 3 activity. As frizzled-related protein (FRZB) is upregulated in LGMD2A muscle samples, it could be hypothesized that beta-catenin regulation is also altered at the Wnt signaling pathway, leading to an incorrect myogenesis. Conversely, expression of most transcription factor genes was downregulated (MYC, FOS and EGR1). Finally, the upregulation of IL-32 and immunoglobulin genes may induce the eosinophil chemoattraction explaining the inflammatory findings observed in presymptomatic stages. The obtained results try to shed some light on identification of novel therapeutic targets for limb-girdle muscular dystrophies.