Epithelial Distribution of E-Cadherin, p63, and Mitotic Figures in ApoTome Images to Determine the Oncogenic Potentiality of Oral Submucous Fibrosis

Nag, Reetoja; Paul, Ranjan Rashmi; Pal, Mousumi; Chatterjee, Jyotirmoy; Das, Raunak Kumar

doi:10.1017/s1431927620024538

Cited by 4 publications

(2 citation statements)

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Section: Resultsmentioning

confidence: 99%

“…Since the TEM data showed that the vesicles in FN mutants are covered by ribosomes and appear to be arising from the RER, we next tested a series of ER and intracellular vesicle and organelle markers in the MSCs using super resolution structured illumination microscopy (23,24) We first analyzed the abundant ER chaperone BIP/GRP78, a member of the heat shock protein 70 family, which assists in protein folding and mediates retention of misfolded proteins (25,26). In the control MSCs we observed a punctate BIP staining throughout the cell cytoplasm (Figure 5A).…”

Section: Misfolded Mutant Fn Is Exported Directly From Rough Endoplas...mentioning

confidence: 99%

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Mutations in Fibronectin Dysregulate Chondrogenesis in Skeletal Dysplasia

Dinesh,

Rousseau,

Mosher

et al. 2023

Preprint

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Fibronectin (FN) is a key extracellular matrix glycoprotein indispensable for the development and function of major vertebrate organ systems. Autosomal dominant FN mutations cause corner fracture-type spondylometaphyseal dysplasia (SMDCF). However, the molecular pathomechanisms underlying this pathology are not known. Here, we have employed patient-derived induced pluripotent stem cells (iPSCs) as an SMDCF cell culture model to investigate the consequences of FN mutations in mesenchymal stem cells (MSCs) and differentiated cartilage-producing chondrocytes. The FN mutations impaired protein secretion from MSCs leading to significant accumulation of FN within the cells and strong reduction of FN in the extracellular matrix. Analyses of plasma samples from SMDCF patients showed a similar reduction in circulating FN. In mutant MSCs, FN accumulated in ribosome-covered intracellular vesicles originating from the rough endoplasmic reticulum and transitioning into lysosomes. These lysosomal vesicles were not cleared from the cytosol. Accumulation of intracellular FN elevated cellular stress markers and altered mitochondrial structure. Bulk RNA sequencing analysis revealed a specific transcriptomic dysregulation of the patient cells relative to controls. Analysis of MSC differentiation into chondrocytes showed impaired mesenchymal condensation for the mutant cells with significant reduction of key chondrogenic markers and impaired cell proliferation. FN mutant cells also displayed alterations in FN splice variants under chondrogenic stimuli. Additionally, FN mutant cells showed significantly downregulated expression of transforming growth factor beta-1 (TGFβ1), a key chondrogenic factor essential for mesenchymal condensation. Exogenous supplementation of FN or TGFβ1 promoted MSC condensation in the mutant cells. Together, the data demonstrate the cellular consequences of SMDCF-causing FN mutations on stem cells and the molecular pathogenic pathways leading to altered chondrogenesis.Significance /HighlightsSMDCF-causing mutations in fibronectin impair protein secretion in iPSC-derived mesenchymal stem cells.Mutant fibronectin and ER protein folding chaperones are directly exported from the rough endoplasmic reticulum into vesicles covered with ribosomes, which transition into lysosomes.These vesicles massively accumulate within the cytosol.Mutations in fibronectin impair mesenchymal condensation of MSCs and their differentiation into chondrocytes.Exogenous addition of purified fibronectin or TGFβ-1 improves mesenchymal condensation of the mutant stem cells.

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Section: Resultsmentioning

confidence: 99%

Section: Misfolded Mutant Fn Is Exported Directly From Rough Endoplas...mentioning

confidence: 99%

Mutations in Fibronectin Dysregulate Chondrogenesis in Skeletal Dysplasia

Dinesh,

Rousseau,

Mosher

et al. 2023

Preprint

View full text Add to dashboard Cite

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Mutations in fibronectin dysregulate chondrogenesis in skeletal dysplasia

Dinesh,

Rousseau,

Mosher

et al. 2024

Cell. Mol. Life Sci.

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Fibronectin (FN) is an extracellular matrix glycoprotein essential for the development and function of major vertebrate organ systems. Mutations in FN result in an autosomal dominant skeletal dysplasia termed corner fracture-type spondylometaphyseal dysplasia (SMDCF). The precise pathomechanisms through which mutant FN induces impaired skeletal development remain elusive. Here, we have generated patient-derived induced pluripotent stem cells as a cell culture model for SMDCF to investigate the consequences of FN mutations on mesenchymal stem cells (MSCs) and their differentiation into cartilage-producing chondrocytes. In line with our previous data, FN mutations disrupted protein secretion from MSCs, causing a notable increase in intracellular FN and a significant decrease in extracellular FN levels. Analyses of plasma samples from SMDCF patients also showed reduced FN in circulation. FN and endoplasmic reticulum (ER) protein folding chaperones (BIP, HSP47) accumulated in MSCs within ribosome-covered cytosolic vesicles that emerged from the ER. Massive amounts of these vesicles were not cleared from the cytosol, and a smaller subset showed the presence of lysosomal markers. The accumulation of intracellular FN and ER proteins elevated cellular stress markers and altered mitochondrial structure. Bulk RNA sequencing revealed a specific transcriptomic dysregulation of the patient-derived cells relative to controls. Analysis of MSC differentiation into chondrocytes showed impaired mesenchymal condensation, reduced chondrogenic markers, and compromised cell proliferation in mutant cells. Moreover, FN mutant cells exhibited significantly lower transforming growth factor beta-1 (TGFβ1) expression, crucial for mesenchymal condensation. Exogenous FN or TGFβ1 supplementation effectively improved the MSC condensation and promoted chondrogenesis in FN mutant cells. These findings demonstrate the cellular consequences of FN mutations in SMDCF and explain the molecular pathways involved in the associated altered chondrogenesis. Supplementary Information The online version contains supplementary material available at 10.1007/s00018-024-05444-4.

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