Dinesh, N. E. H. | bioRxiv Science

bioRxiv2024

Role of Fibronectin in Postnatal Skeletal Development

Fibronectin (FN) is a ubiquitous matrix glycoprotein essential for the physiological development of various tissues. Mutations in FN cause a form of skeletal dysplasia, emphasizing the importance of FN in cartilage and bone development. However, the relevance and functional role of FN during skeletal development remains elusive. We employed conditional knockout mouse models for the cellular FN isoform in cartilage (cFNKO), the plasma FN isoform in hepatocytes (pFNKO), and a double knockout (FNdKO) to determine the relevance of these two principal FN isoforms in postnatal skeletal development spanning from P1 to 2 months of age. We identified a unique topological FN deposition pattern in the mouse limb with prominent levels at the resting and hypertrophic chondrocyte zones and in the trabecular bone. Circulating pFN did not enter the growth plate and was limited to the primary ossification center, whereas cartilage-specific cFN was detected as the major isoform in epiphyseal cartilage. Deletion of either one of the isoforms in single knockouts (cFNKO or pFNKO) only led to subtle changes in some of the analyzed parameters. Complete ablation of both cFN in the growth plate and circulating pFN in plasma resulted in significantly reduced postnatal body weight, body length, and bone length in the FNdKO mice. Assessment of the FNdKO adult bone microarchitecture using micro-CT revealed significantly reduced trabecular bone volume, trabecular network, bone mineral density, and increased bone marrow adiposity. Analysis of chondrogenesis in FNdKO mice showed changes in the proliferating and hypertrophic growth plate zones, consistent alterations in chondrogenic markers such as collagen type II and type X, reduced apoptosis of hypertrophic chondrocytes, and downregulation of bone formation markers. FNdKO mice also displayed decreased levels of transforming growth factor-{beta}1 (TGF{beta}1) and downstream phospho-AKT levels, which are critical regulators of chondrogenesis and bone formation. In conclusion, the data demonstrate that FN is essential for proper chondrogenesis and postnatal bone development. Simultaneous deletion of both FN isoforms in the developing cartilage leads to critical TGF{beta}-mediated alterations in chondrogenic differentiation, resulting in bone and skeletal defects. Significance/HighlightsO_LIFN is highly expressed during mouse limb development with increased deposition in resting and hypertrophic chondrocyte zones and the primary ossification center. C_LIO_LICartilage-specific cFN and circulating pFN are distinctly distributed during embryonic and postnatal bone development, with chondrocyte-specific cFN present in the growth plate and pFN limited to trabecular bone and the bone marrow. C_LIO_LIDeletion of both cFN and pFN leads to reduced bone growth during early postnatal development. C_LIO_LIDeletion of cFN and pFN leads to reduced trabecular bone formation, bone mineralization, and increased bone marrow adiposity in 2-month adult mice. C_LIO_LIAbsence of both FN isoforms in the FNdKO mouse model leads to altered chondrogenesis and reduced bone formation. C_LIO_LIFN regulates chondrogenesis via TGF{beta}-mediated phospho-AKT signaling. C_LI

developmental biology↗

bioRxiv2023

Mutations in Fibronectin Dysregulate Chondrogenesis in Corner Fracture Type Spondylometaphyseal Dysplasia

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 and transitioned into lysosomes. Massive amounts of these vesicles were not cleared from the cytosol. 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. FN mutant cells also displayed altered FN splice variants under chondrogenic stimuli. Moreover, FN mutant cells exhibited significantly lower transforming growth factor beta-1 (TGF{beta}1) expression, crucial for mesenchymal condensation. Exogenous FN or TGF{beta}1 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. Significance /Highlights* SMDCF-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. * The cells cannot clear the massive accumulation of cytosolic vesicles. * Mutations in fibronectin impair stem cell proliferation, mesenchymal condensation, and the differentiation of MSCs into chondrocytes. * Exogenous addition of purified fibronectin or TGF{beta}-1 improves mesenchymal condensation and chondrogenesis of the FN mutant stem cells.

cell biology↗