Gingival proteomics reveals the role of TGF beta and YAP/TAZ signaling in Raine syndrome fibrosis.

Raine syndrome (RNS) is a rare autosomal recessive osteosclerotic dysplasia. RNS is caused by loss-of-function disease-causative variants of the FAM20C gene that encodes a kinase that phosphorylates most of the secreted proteins found in the body fluids and extracellular matrix. The most common RNS clinical features are generalized osteosclerosis, facial dysmorphism, intracerebral calcifications and respiratory defects. In non-lethal RNS forms, oral traits include a well-studied hypoplastic amelogenesis imperfecta (AI) and a much less characterized gingival phenotype. We used immunomorphological, biochemical, and siRNA approaches to analyze gingival tissues and primary cultures of gingival fibroblasts of two unrelated, previously reported RNS patients. We showed that fibrosis, pathological gingival calcifications and increased expression of various profibrotic and pro-osteogenic proteins such as POSTN, SPARC and VIM were common findings. Proteomic analysis of differentially expressed proteins demonstrated that proteins involved in extracellular matrix (ECM) regulation and related to the TGFß/SMAD signaling pathway were increased. Functional analyses confirmed the upregulation of TGFß/SMAD signaling and subsequently uncovered the involvement of two closely related transcription cofactors important in fibrogenesis, Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ). Knocking down of FAM20C confirmed the TGFß-YAP/TAZ interplay indicating that a profibrotic loop enabled gingival fibrosis in RNS patients. In summary, our in vivo and in vitro data provide a detailed description of the RNS gingival phenotype. They show that gingival fibrosis and calcifications are associated with, and most likely caused by excessed ECM production and disorganization. They furthermore uncover the contribution of increased TGFß-YAP/TAZ signaling in the pathogenesis of the gingival fibrosis.

Enamel and dentin in Enamel renal syndrome: A confocal Raman microscopy view.

Enamel Renal Syndrome (ERS) is a rare genetic disorder caused by biallelic mutations in Family with sequence similarity 20A (FAM20A) gene encoding the secretory pathway pseudokinase FAM20A. ERS is characterized by hypoplastic amelogenesis imperfecta (AI), impaired tooth eruption, intra-pulpal calcifications, gingival fibromatosis and nephrocalcinosis of various severity. Previous studies showed that the hypoplastic enamel was also hypomineralized but its chemical composition has not been extensively studied. Furthermore it is currently unclear whether dentinal defects are associated with AI in ERS patients. The objective of the study was to provide a structural and chemical analysis of enamel, dentin and dentin enamel junction (DEJ) in ERS patients carrying four, previously reported, distinct mutations in FAM20A. Chemical cartography obtained with Raman microscopy showed that compared to control samples, ERS enamel composition was severely altered and a cementum-like structure was observed in some cases. Chemical composition of peripulpal dentin was also affected and usual gradient of phosphate intensity, shown in DEJ profile, was absent in ERS samples. DEJ and dentinal anomalies were further confirmed by scanning electron microscopy analysis. In conclusion, our study shows that enamel formation is severely compromised in ERS patients and provides evidence that dentinal defects are an additional feature of the ERS dental phenotype.

Cubilin, the intrinsic factor-vitamin B12 receptor.

Cubilin (CUBN), the intrinsic factor-vitamin B12 receptor is a large endocytic protein involved in various physiological functions: vitamin B12 uptake in the gut; reabsorption of albumin and maturation of vitamin D in the kidney; nutrient delivery during embryonic development. Cubilin is an atypical receptor, peripherally associated to the plasma membrane. The transmembrane proteins amnionless (AMN) and Lrp2/Megalin are the currently known molecular partners contributing to plasma membrane transport and internalization of Cubilin. The role of Cubilin/Amn complex in the handling of vitamin B12 in health and disease has extensively been studied and so is the role of the Cubilin-Lrp2 tandem in renal pathophysiology. Accumulating evidence strongly supports a role of Cubilin in some developmental defects including impaired closure of the neural tube. Are these defects primarily caused by the dysfunction of a specific Cubilin ligand or are they secondary to impaired vitamin B12 or protein uptake? We will present the established Cubilin functions, discuss the developmental data and provide an overview of the emerging implications of Cubilin in the field of cardiovascular disease and cancer pathogenesis.

Pathogenesis of Enamel-Renal Syndrome Associated Gingival Fibromatosis: A Proteomic Approach.

The enamel renal syndrome (ERS) is a rare disorder featured by amelogenesis imperfecta, gingival fibromatosis and nephrocalcinosis. ERS is caused by bi-allelic mutations in the secretory pathway pseudokinase FAM20A. How mutations in FAM20A may modify the gingival connective tissue homeostasis and cause fibromatosis is currently unknown. We here analyzed conditioned media of gingival fibroblasts (GFs) obtained from four unrelated ERS patients carrying distinct mutations and control subjects. Secretomic analysis identified 109 dysregulated proteins whose abundance had increased (69 proteins) or decreased (40 proteins) at least 1.5-fold compared to control GFs. Proteins over-represented were mainly involved in extracellular matrix organization, collagen fibril assembly, and biomineralization whereas those under-represented were extracellular matrix-associated proteins. More specifically, transforming growth factor-beta 2, a member of the TGFß family involved in both mineralization and fibrosis was strongly increased in samples from GFs of ERS patients and so were various known targets of the TGFß signaling pathway including Collagens, Matrix metallopeptidase 2 and Fibronectin. For the over-expressed proteins quantitative RT-PCR analysis showed increased transcript levels, suggesting increased synthesis and this was further confirmed at the tissue level. Additional immunohistochemical and western blot analyses showed activation and nuclear localization of the classical TGFß effector phospho-Smad3 in both ERS gingival tissue and ERS GFs. Exposure of the mutant cells to TGFB1 further upregulated the expression of TGFß targets suggesting that this pathway could be a central player in the pathogenesis of the ERS gingival fibromatosis. In conclusion our data strongly suggest that TGFß -induced modifications of the extracellular matrix contribute to the pathogenesis of ERS. To our knowledge this is the first proteomic-based analysis of FAM20A-associated modifications.

Cubilin, the Intrinsic Factor-Vitamin B12 Receptor in Development and Disease.

Gp280/Intrinsic factor-vitamin B12 receptor/Cubilin (CUBN) is a large endocytic receptor serving multiple functions in vitamin B12 homeostasis, renal reabsorption of protein or toxic substances including albumin, vitamin D-binding protein or cadmium. Cubilin is a peripheral membrane protein consisting of 8 Epidermal Growth Factor (EGF)-like repeats and 27 CUB (defined as Complement C1r/C1s, Uegf, BMP1) domains. This structurally unique protein interacts with at least two molecular partners, Amnionless (AMN) and Lrp2/Megalin. AMN is involved in appropriate plasma membrane transport of Cubilin whereas Lrp2 is essential for efficient internalization of Cubilin and its ligands. Observations gleaned from animal models with Cubn deficiency or human diseases demonstrate the importance of this protein. In this review addressed to basic research and medical scientists, we summarize currently available data on Cubilin and its implication in renal and intestinal biology. We also discuss the role of Cubilin as a modulator of Fgf8 signaling during embryonic development and propose that the Cubilin-Fgf8 interaction may be relevant in human pathology, including in cancer progression, heart or neural tube defects. We finally provide experimental elements suggesting that some aspects of Cubilin physiology might be relevant in drug design.

Preliminary study of the safety and efficacy of medium-chain triglycerides for use as an intraocular tamponading agent in minipigs.

PURPOSE: To date, only silicone oils and gases have the appropriate characteristics for use in vitreo-retinal surgery as vitreous substitutes with intraocular tamponading properties. This preliminary study evaluated the safety and efficacy of medium-chain triglycerides (MCTs) for use as a tamponading agent in minipigs. METHODS: In 15 minipigs, 15 right eyes underwent vitrectomies followed by injection of MCT tamponade (day 1). Two groups were defined. In Group A (ten eyes), the surgical procedure before MCT injection included induced rhegmatogenous retinal detachment (RRD), retina flattening, and retinopexy. In Group B (five eyes), MCT was injected without inducing RRD; in these eyes, MCT was removed on day 90. Pigs were sacrificed on day 45 (Group A) or 120 (Group B). Eyes were examined on days 1, 5, 15, and 45 in both groups and on days 90 and 120 in Group B. In Group B only, we performed bilateral electroretinography examinations on days 1 and 120, and histological examinations of MCTs and controlateral eyes were performed after sacrifice. RESULTS: In Group A eyes (n = 9; one eye was non-assessable), on day 45, the retina was flat in seven eyes and two RRD detachments were observed in insufficiently MCT-filled eyes. In Group B, electroretinography showed no significant differences between MCT eyes and controls on days 1 or 120. Histological analyses revealed no signs of retinal toxicity. CONCLUSIONS: This study showed that MCT tamponade seems to be effective and safe; however, additional studies are needed before it becomes commonly used as a tamponading agent in humans.

Megalin mediates the influence of sonic hedgehog on oligodendrocyte precursor cell migration and proliferation during development.

Oligodendrocyte precursor cells (OPCs) of the optic nerve are generated in the preoptic area, from where they migrate to colonize it entirely. Sonic hedgehog (Shh) induces the proliferation of these cells as well as influencing their migration, acting through its canonical receptor (Ptc-1). However, the multiligand receptor megalin (or LRP-2) is also involved in Shh-induced OPC proliferation and migration, and thus, we have evaluated the relevance of this interaction. During the stages at which Shh influences OPC development, we found megalin to be selectively expressed by optic nerve astrocytes, whereas Ptc-1 and Gli1 were found in OPCs. Indeed, this pattern of expression paralleled the rostral-caudal expression of the three Shh-related molecules during the time course of plp-dm20(+) -OPC colonization. The blockage of megalin partially abolished OPC chemoattraction and fully impaired Shh-induced proliferation. Using in vitro co-cultures of dissociated optic nerve cells, we demonstrated that Shh was internalized by astrocytes via megalin, and sufficient Shh was subsequently released to produce the biological effects on OPCs observed in the nerve. Together, these data indicate that at least part of the influence of Shh on OPCs is mediated by megalin during optic nerve development, and that astrocytes expressing megalin transiently capture Shh to present it to OPCs and/or to control the gradient of this molecule during development.

Developmental cell death is enhanced in the cerebral cortex of mice lacking the brain vesicular monoamine transporter.

Neurotransmitters have emerged as important players in the control of programmed cell death in the cerebral cortex. We report that genetic depletion of serotonin, dopamine, and norepinephrine in mice lacking the vesicular monoamine transporter (VMAT2 KO mice) causes an increase in cell death in the superficial layers of the cingulate and retrosplenial cortices during early postnatal life (postnatal days 0-4). Electron microscopy and terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling indicated that this represents a form of apoptosis. Caspase-3 and -9 are over activated in the VMAT2 KO cortex and Bcl-X(L) is downregulated, whereas the apoptosis-inducing factor caspase-8 and FasL/FasR pathway are not involved. Partial inhibition of serotonin or/and catecholamine synthesis by pharmacological treatments or genetic reduction of serotonin neuron number in mice lacking the transcription factor Pet-1 (pheochromocytoma 12 E26 transformation-specific) did not modify the cell death ratios in the cerebral cortex. However, when monoamine oxidase type A was invalidated in the VMAT2 KO background (VMAT2-MAOA DKO mice), increases in 5-HT levels coincided with a reduction of cell death and a normalization of Bcl-X(L) expression. trkB signaling is not implicated in the anti-apoptotic effects of MAOA inhibition because BDNF mRNA levels were unchanged in VMAT2-MAOA DKO mice and because the massive cell death in the cerebral cortex of trkB KO mice is also reverted by genetic invalidation of the MAOA gene. Finally the broad 5-HT2 receptor agonist (-)-2,5-dimethoxy-4-iodoamphetamine hydrochloride prevented the increase in cell death of VMAT2 KO mice. Altogether, these results suggest that high levels of serotonin, acting through 5-HT2 receptors, have neuroprotective action on cortical neurons by controlling Bcl-X(L) mRNA levels and that this action is independent of trkB signaling.

Biochemical characterization of the mammalian Cux2 protein.

The CCAAT displacement protein (CDP) and cux (Cut homeobox) genes were originally identified as the human and mouse orthologs of Drosophila melanogaster cut. More recently, vertebrates were found to possess a second cut orthologs that was generated by gene duplication: Cux2. We report the initial biochemical characterization of the Cux2 protein in tissue culture and in vitro. We generated four polyclonal antibodies that were able to recognize the human and mouse Cux2 protein but displayed little or no cross-reactivity towards CDP1 and Cux1. The expression of the Cux2 protein was convincingly detected in only one among 19 neuronal cell lines: the SH-SY5Y human neuroblastoma cell line. CDP/Cux proteins contain four DNA binding domains, three Cut repeat (CR1, CR2 and CR3) and one Cut homeodomain (HD). Purified fusion proteins containing either CR1CR2, CR2CR3HD or CR3HD exhibited similar DNA binding specificities as the corresponding domains of Cux1, but their DNA binding kinetics were much more rapid. Similarly, the full-length Cux2 protein made rapid but transient interactions with DNA. We did not observe an N-terminally processed Cux2 isoform equivalent to the Cux1 p110 isoform. Whereas Cux1 can function as a repressor or activator in a promoter-specific manner, Cux2 functioned exclusively as a transcriptional repressor in NIH3T3 cells. Overall, our results suggest that the Cux1 and Cux2 proteins carry distinct biochemical functions. Cux2 is able, like Cux1, to perform the CCAAT-displacement activity. However, Cux2 is unlikely to execute transcriptional regulatory functions that require stable interaction with DNA.