Ablation of the miRNA cluster 24 in cartilage and osteoblasts impairs bone remodeling.

MicroRNAs (miRNAs) post-transcriptionally regulate cartilage and bone development and function, however, only few miRNAs have been described to play a role for cartilage to bone transition in vivo. Previously, we showed that cartilage-specific deletion of the Mirc24 cluster in newborn male mice leads to impaired growth plate cartilage development due to increased RAF/MEK/ERK signaling and affects the stability of the cartilage extracellular matrix on account of decreased SOX6 and SOX9 and increased MMP13 levels. Here, we studied how Mirc24 cluster inactivation in cartilage and osteoblasts leads to an increased bone density associated with defects in collagen remodeling in trabecular bone. No changes in osteoblast distribution were observed, whereas the number of osteoclasts was reduced and TRAP activity in osteoclasts decreased. Surprisingly, an increased level of cluster-encoded miR-322 or miR-503 raises Rankl gene expression and inactivation of the cluster in chondrocytes reduces Rankl expression. These results suggest that the Mirc24 cluster regulates Rankl expression in chondrocytes at the chondro-osseous border, where the cluster is mainly expressed to modulate osteoclast formation, bone remodeling and bone integrity.

Ablation of the miRNA Cluster 24 Has Profound Effects on Extracellular Matrix Protein Abundance in Cartilage.

MicroRNAs (miRNAs) regulate cartilage differentiation and contribute to the onset and progression of joint degeneration. These small RNA molecules may affect extracellular matrix organization (ECM) in cartilage, but for only a few miRNAs has this role been defined in vivo. Previously, we showed that cartilage-specific genetic ablation of the Mirc24 cluster in mice leads to impaired cartilage development due to increased RAF/MEK/ERK pathway activation. Here, we studied the expression of the cluster in cartilage by LacZ reporter gene assays and determined its role for extracellular matrix homeostasis by proteome and immunoblot analysis. The cluster is expressed in prehypertrophic/hypertrophic chondrocytes of the growth plate and we now show that the cluster is also highly expressed in articular cartilage. Cartilage-specific loss of the cluster leads to increased proteoglycan 4 and matrix metallopeptidase 13 levels and decreased aggrecan and collagen X levels in epiphyseal cartilage. Interestingly, these changes are linked to a decrease in SRY-related HMG box-containing (SOX) transcription factors 6 and 9, which regulate ECM production in chondrocytes. Our data suggests that the Mirc24 cluster is important for ECM homoeostasis and the expression of transcriptional regulators of matrix production in cartilage.

Respiratory chain inactivation links cartilage-mediated growth retardation to mitochondrial diseases.

In childhood, skeletal growth is driven by transient expansion of cartilage in the growth plate. The common belief is that energy production in this hypoxic tissue mainly relies on anaerobic glycolysis and not on mitochondrial respiratory chain (RC) activity. However, children with mitochondrial diseases causing RC dysfunction often present with short stature, which indicates that RC activity may be essential for cartilage-mediated skeletal growth. To elucidate the role of the mitochondrial RC in cartilage growth and pathology, we generated mice with impaired RC function in cartilage. These mice develop normally until birth, but their later growth is retarded. A detailed molecular analysis revealed that metabolic signaling and extracellular matrix formation is disturbed and induces cell death at the cartilage-bone junction to cause a chondrodysplasia-like phenotype. Hence, the results demonstrate the overall importance of the metabolic switch from fetal glycolysis to postnatal RC activation in growth plate cartilage and explain why RC dysfunction can cause short stature in children with mitochondrial diseases.

Depletion of Collagen IX Alpha1 Impairs Myeloid Cell Function.

The trabecular extracellular matrix (ECM) forms a three-dimensional scaffold to stabilize the bone marrow, provide substrates for cell-matrix interactions and retain, present or release signals to modulate hematopoietic stem and progenitor cell development. However, the impact of trabecular ECM components on hematopoiesis has been poorly studied. Using collagen IX alpha1 - deficient (Col9a1(-/-) ) mice, we revealed that a lack of collagen IX alpha1 results in a disorganized trabecular network enriched in fibronectin, and in a reduction in myeloid cells, which was accompanied by a decrease in colony-stimulating factor 1 receptor expression on monocytes from the bone marrow. In contrast, B-cell numbers in the bone marrow and T-cell numbers in the thymus remained unchanged. Alterations in the bone marrow microenvironment may not only reduce myeloid cell numbers, but also have long-term implications for myeloid cell function. Mice were infected with Listeria moncytogenes to analyze the function of myeloid cells. In this case, an inadequate macrophage-dependent clearance of bacterial infections was observed in Col9a1(-/-) mice in vivo. This was mainly caused by an impaired interferon-gamma/tumor necrosis factor-alpha-mediated activation of macrophages. The loss of collagen IX alpha1 therefore destabilizes the trabecular bone network, impairs myeloid cell differentiation, and affects the innate immune response against Listeria. Stem Cells 2018;36:1752-1763.

miR-322 stabilizes MEK1 expression to inhibit RAF/MEK/ERK pathway activation in cartilage.

Cartilage originates from mesenchymal cell condensations that differentiate into chondrocytes of transient growth plate cartilage or permanent cartilage of the articular joint surface and trachea. MicroRNAs fine-tune the activation of entire signaling networks and thereby modulate complex cellular responses, but so far only limited data are available on miRNAs that regulate cartilage development. Here, we characterize a miRNA that promotes the biosynthesis of a key component in the RAF/MEK/ERK pathway in cartilage. Specifically, by transcriptome profiling we identified miR-322 to be upregulated during chondrocyte differentiation. Among the various miR-322 target genes in the RAF/MEK/ERK pathway, only Mek1 was identified as a regulated target in chondrocytes. Surprisingly, an increased concentration of miR-322 stabilizes Mek1 mRNA to raise protein levels and dampen ERK1/2 phosphorylation, while cartilage-specific inactivation of miR322 in mice linked the loss of miR-322 to decreased MEK1 levels and to increased RAF/MEK/ERK pathway activation. Such mice died perinatally due to tracheal growth restriction and respiratory failure. Hence, a single miRNA can stimulate the production of an inhibitory component of a central signaling pathway to impair cartilage development.

SorCS1 variants and amyloid precursor protein (APP) are co-transported in neurons but only SorCS1c modulates anterograde APP transport.

Processing of amyloid precursor protein (APP) into amyloid-ß peptide (Aß) is crucial for the development of Alzheimer's disease (AD). Because this processing is highly dependent on its intracellular itinerary, altered subcellular targeting of APP is thought to directly affect the degree to which Aß is generated. The sorting receptor SorCS1 has been genetically linked to AD, but the underlying molecular mechanisms are poorly understood. We analyze two SorCS1 variants; one, SorCS1c, conveys internalization of surface-bound ligands whereas the other, SorCS1b, does not. In agreement with previous studies, we demonstrate co-immunoprecipitation and co-localization of both SorCS1 variants with APP. Our results suggest that SorCS1c and APP are internalized independently, although they mostly share a common post-endocytic pathway. We introduce functional Venus-tagged constructs to study SorCS1b and SorCS1c in living cells. Both variants are transported by fast anterograde axonal transport machinery and about 30% of anterograde APP-positive transport vesicles contain SorCS1. Co-expression of SorCS1b caused no change of APP transport kinetics, but SorCS1c reduced the anterograde transport rate of APP and increased the number of APP-positive stationary vesicles. These data suggest that SorCS1 and APP share trafficking pathways and that SorCS1c can retain APP from insertion into anterograde transport vesicles. Altered APP trafficking is thought to modulate its processing. SorCS1 has been suggested to function in APP trafficking. We analyzed if the two SorCS1 variants, SorCS1b and SorCS1c, tie APP to the cell surface or modify its internalization and intracellular targeting. We observed co-localization and vesicular co-transport of APP and SorCS1, but independent internalization and sorting through a common post-endocytic pathway. Co-expression of one variant, SorCS1c, reduced anterograde APP transport. These data demonstrate that SorCS1 and APP share trafficking pathways and that SorCS1c can retain APP from insertion into anterograde transport vesicles.

MiR-26a modulates extracellular matrix homeostasis in cartilage.

MicroRNAs (miRNAs) may represent new therapeutic targets for bone and joint diseases. We hypothesized that several cartilage-specific proteins are targeted by a single miRNA and used bioinformatics to identify a miRNA that can modulate extracellular matrix (ECM) homeostasis in cartilage. Bioinformatic analysis of miRNA binding sequences in the 3'-untranslated region (3'-UTR) of target genes was performed to identify a miRNA that could bind to the 3'-UTR of cartilage matrix-related genes. MiRNA expression was studied by quantitative PCR of microdissected growth plate cartilage and binding to the 3'-UTR sequences was analyzed by luciferase interaction studies. Levels of proteins encoded by target genes in cultures of miR-26a mimic- or inhibitor-transfected chondrocytes were determined by FACS or immunoblot analysis. The complementary binding sequence of miR-26a and miR-26b was found in the 3'-UTR of the prehypertrophic/hypertrophic-specific genes Cd200, Col10a1 as well as Col9a1 and Ctgf. Both miRNAs were expressed in cartilage and only miR-26a was downregulated in hypertrophic growth plate cartilage. MiR-26a could interact with the 3'-UTR of Cd200 and Col10a1 in luciferase binding studies, but not with Col9a1 and Ctgf. However, protein expression of target genes and the ECM adaptor genes matrilin-3 and COMP was significantly altered in miR-26a mimic- or inhibitor-transfected chondrocytes, whereas the abundance of the cell surface receptor for insulin was not changed. In conclusion, miR-26a suppresses hypertrophic and ECM adaptor protein production. Dysregulation of miR-26a expression could contribute to ECM changes in cartilage diseases and this miRNA may therefore act as a therapeutic target.

Normal cerebellar development in S100B-deficient mice.

The calcium-binding protein S100B has been shown to support neuron proliferation, migration and neurite growth in vitro, while the significance of S100B for neuronal development in vivo is controversial. We have investigated the effect of S100B deficiency on cerebellar development in S100B knockout mice at an age of 5 and 10 days after birth (P5 and P10). This time range covers important developmental steps in the cerebellum such as granule cell proliferation and migration, as well as dendritic growth of Purkinje cells. Bergmann glial cells contain a particularly high concentration of S100B and serve as scaffold for both migrating granule cells and growing Purkinje cell dendrites. This renders the postnatal cerebellum ideal as a model system to study the importance of S100B for glial and neuronal development. We measured the length of Bergmann glial processes, the width of the external granule cell layer as a measure of granule cell proliferation, the decrease in width of the external granule cell layer between P5 and P10 as a measure of granule cell migration, and the length of Purkinje cell dendrites in wild-type and S100B knockout mice. None of these parameters showed significant differences between wild-type and knockout mice. In addition, wild-type and knockout mice performed equally in locomotor behaviour tests. The results indicate that S100B-deficient mice have normal development of the cerebellum and no severe impairment of motor function.