Dominant missense variants in SREBF2 are associated with complex dermatological, neurological, and skeletal abnormalities.

PURPOSE: We identified 2 individuals with de novo variants in SREBF2 that disrupt a conserved site 1 protease (S1P) cleavage motif required for processing SREBP2 into its mature transcription factor. These individuals exhibit complex phenotypic manifestations that partially overlap with sterol regulatory element binding proteins (SREBP) pathway-related disease phenotypes, but SREBF2-related disease has not been previously reported. Thus, we set out to assess the effects of SREBF2 variants on SREBP pathway activation. METHODS: We undertook ultrastructure and gene expression analyses using fibroblasts from an affected individual and utilized a fly model of lipid droplet (LD) formation to investigate the consequences of SREBF2 variants on SREBP pathway function. RESULTS: We observed reduced LD formation, endoplasmic reticulum expansion, accumulation of aberrant lysosomes, and deficits in SREBP2 target gene expression in fibroblasts from an affected individual, indicating that the SREBF2 variant inhibits SREBP pathway activation. Using our fly model, we discovered that SREBF2 variants fail to induce LD production and act in a dominant-negative manner, which can be rescued by overexpression of S1P. CONCLUSION: Taken together, these data reveal a mechanism by which SREBF2 pathogenic variants that disrupt the S1P cleavage motif cause disease via dominant-negative antagonism of S1P, limiting the cleavage of S1P targets, including SREBP1 and SREBP2.

Spatial transcriptomics implicates impaired BMP signaling in NF1 fracture pseudarthrosis in murine and patient tissues.

The neurofibromatosis type 1 (NF1) RASopathy is associated with persistent fibrotic nonunions (pseudarthrosis) in human and mouse skeletal tissue. Here, we performed spatial transcriptomics to define the molecular signatures occurring during normal endochondral healing following fracture in mice. Within the control fracture callus, we observed spatially restricted activation of morphogenetic pathways, such as TGF-ß, WNT, and BMP. To investigate the molecular mechanisms contributing to Nf1-deficient delayed fracture healing, we performed spatial transcriptomic analysis on a Postn-cre;Nf1fl/- (Nf1Postn) fracture callus. Transcriptional analyses, subsequently confirmed through phospho-SMAD1/5/8 immunohistochemistry, demonstrated a lack of BMP pathway induction in Nf1Postn mice. To gain further insight into the human condition, we performed spatial transcriptomic analysis of fracture pseudarthrosis tissue from a patient with NF1. Analyses detected increased MAPK signaling at the fibrocartilaginous-osseus junction. Similar to that in the Nf1Postn fracture, BMP pathway activation was absent within the pseudarthrosis tissue. Our results demonstrate the feasibility of delineating the molecular and tissue-specific heterogeneity inherent in complex regenerative processes, such as fracture healing, and reconstructing phase transitions representing endochondral bone formation in vivo. Furthermore, our results provide in situ molecular evidence of impaired BMP signaling underlying NF1 pseudarthrosis, potentially informing the clinical relevance of off-label BMP2 as a therapeutic intervention.

Molecular Dissection of Somatic Skeletal Disease in Neurofibromatosis Type 1.

Neurofibromatosis type 1 (NF1) is a tumor predisposition syndrome caused by heterozygous NF1 gene mutations. Patients with NF1 present with pleiotropic somatic secondary manifestations, including development of bone pseudarthrosis after fracture. Somatic NF1 gene mutations were reproducibly identified in patient-derived pseudarthrosis specimens, suggesting a local mosaic cell population including somatic pathologic cells. The somatic cellular pathogenesis of NF1 pseudarthroses remains unclear, though defects in osteogenesis have been posited. Here, we applied time-series single-cell RNA-sequencing (scRNA-seq) to patient-matched control and pseudarthrosis-derived primary bone stromal cells (BSCs). We show that osteogenic specification to an osteoblast progenitor cell population was evident for control bone-derived cells and haploinsufficient pseudarthrosis-derived cells. Similar results were observed for somatic patient fracture-derived NF1-/- cells; however, expression of genetic pathways associated with skeletal mineralization were significantly reduced in NF1-/- cells compared with fracture-derived NF1+/- cells. In mice, we show that Nf1 expressed in bone marrow osteoprogenitors is required for the maintenance of the adult skeleton. Results from our study implicate impaired Clec11a-Itga11-Wnt signaling in the pathogenesis of NF1-associated skeletal disease. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Neurofibromin deficiency-associated transcriptional dysregulation suggests a novel therapy for tibial pseudoarthrosis in NF1.

Neurofibromatosis type 1 (NF1) is an autosomal dominant disease caused by mutations in NF1. Among the earliest manifestations is tibial pseudoarthrosis and persistent nonunion after fracture. To further understand the pathogenesis of pseudoarthrosis and the underlying bone remodeling defect, pseudoarthrosis tissue and cells cultured from surgically resected pseudoarthrosis tissue from NF1 individuals were analyzed using whole-exome and whole-transcriptome sequencing as well as genomewide microarray analysis. Genomewide analysis identified multiple genetic mechanisms resulting in somatic biallelic NF1 inactivation; no other genes with recurring somatic mutations were identified. Gene expression profiling identified dysregulated pathways associated with neurofibromin deficiency, including phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling pathways. Unlike aggressive NF1-associated malignancies, tibial pseudoarthrosis tissue does not harbor a high frequency of somatic mutations in oncogenes or other tumor-suppressor genes, such as p53. However, gene expression profiling indicates that pseudoarthrosis tissue has a tumor-promoting transcriptional pattern, despite lacking tumorigenic somatic mutations. Significant overexpression of specific cancer-associated genes in pseudoarthrosis highlights a potential for receptor tyrosine kinase inhibitors to target neurofibromin-deficient pseudoarthrosis and promote proper bone remodeling and fracture healing.

High-concentration of BMP2 reduces cell proliferation and increases apoptosis via DKK1 and SOST in human primary periosteal cells.

BMP2, a well-known osteoinductive agent approved by FDA, is currently being used for various off-label orthopedic applications. Recently, concerns about its efficacy for off-label use, concentration, and complications have emerged. Interestingly, there is an extremely large discrepancy in BMP2 concentration between clinical use (i.e. 1.5mg/ml) and in vitro studies (50-300 ng/ml). The purpose of this study was to determine the effects of a relatively high-concentration of BMP2 on cell proliferation and apoptosis using human primary periosteal cells as BMP2 is generally applied around the periosteum in orthopedic surgeries. We isolated periosteal cells from three independent patients. The cell proliferation assessed by MTT activity was significantly reduced by a high-concentration of BMP2 (~2000 ng/ml), while such a reduction was not observed by using a low-concentration of BMP2 (~200 ng/ml). The cell apoptosis assessed by caspase activity was significantly increased by high-concentration BMP2, while such an increase was not observed by low-concentration BMP2. We found that Wnt signaling activity was significantly reduced by high-concentration BMP2 along with a dramatic increase in DKK1 and SOST, key inhibitors of Wnt signaling in bone. The addition of DKK1 or SOST protein to the primary periosteal cells reduced MTT activity and significantly increased caspase activity. Silencing the DKK1 or SOST expression using the siRNA technique normalized cell proliferation and apoptosis in the periosteum-derived cells when exposed to a high-concentration BMP2. Taken together, these results suggest that a high-concentration BMP2 decreases human periosteal cell proliferation and induces apoptosis via the activation of Wnt inhibitors DKK1 and SOST. This study provides new insights to the effects of high BMP2 concentration on human periosteal cells and brings out the possibility of multiple effects of current BMP2 therapy on various skeletal tissues.

Acute BMP2 upregulation following induction of ischemic osteonecrosis in immature femoral head.

Juvenile ischemic osteonecrosis of the femoral head (IOFH) is one of the most serious hip conditions causing the femoral head deformity. Little is known about BMP signaling following ischemic osteonecrosis. In this study, we found acute BMP2 upregulation in the femoral head cartilage 24h after ischemic induction using our immature pig IOFH model. Similarly, in our ischemic osteonecrosis mouse model, BMP2 expression and BMP signaling were enhanced in the articular cartilage surrounding the necrotic bone. BMP2 was increased in cartilage explants and primary chondrocytes under hypoxia (1% O(2)) compared with normoxia (21% O(2)). Addition of the hypoxia inducible factor 1 (HIF1) activator DFO significantly increased BMP2 while HIF1 silencing (siHIF1) only partially reduced BMP2, suggesting other mechanisms of BMP2 upregulation being present. Hypoxia is known to induce the production of free oxygen radicals, which are converted to hydrogen peroxide (H(2)O(2)) by superoxide dismutase 2 (SOD2). As an alternative mechanism, we investigated the effect of H(2)O(2)/SOD2 production on BMP2 upregulation. Chondrocytes produced more H(2)O(2) under hypoxia than normoxia. H(2)O(2) addition to the chondrocyte culture also significantly increased BMP2 expression. SOD2 was also dramatically increased in the ischemic pig cartilage at 24h following surgery and in primary chondrocytes/cartilage explants culture under hypoxia. SOD2 protein addition to the chondrocyte culture significantly increased BMP2. Moreover, DFO significantly increased SOD2 while HIF1 silencing only partially reduced SOD2. These results suggest that the acute BMP2 response of chondrocytes to ischemic osteonecrosis is more dominantly through the H(2)O(2) production and only partly through the HIF1 pathway.

Structure-function relationships in the stereospecific and manganese-dependent 3,4-dihydroxyphenylalanine/tyrosine-sulfating activity of human monoamine-form phenol sulfotransferase, SULT1A3.

The human monoamine-form phenol sulfotransferase (PST), SULT1A3, has a unique 3,4-dihydroxyphenylalanine (Dopa)/tyrosine-sulfating activity that is stereospecific for their d-form enantiomers and can be stimulated dramatically by Mn(2+). This activity is not present in the simple phenol-form PST, SULT1A1, which is otherwise >93% identical to SULT1A3 in amino acid sequence. The majority of the differences between these two proteins reside in two variable regions of their sequences. Through the characterization of chimeric PSTs where these two regions were exchanged between them, it was demonstrated that variable Region II of SULT1A3 is required for the stereospecificity of its Dopa/tyrosine-sulfating activity, whereas variable Region I of SULT1A3 is required for the stimulation by Mn(2+) of this activity. Further studies using point-mutated SULT1A3s mutated at amino acid residues in these two regions and deletional mutants missing residues 84-86 and 84-90 implicate residue Glu-146 (in variable Region II of SULT1A3), as well as the presence of residues 84-90 of variable Region I, in the stereospecificity in the absence of Mn(2+). Residue Asp-86 (in variable Region I of SULT1A3), on the other hand, is critical in the Mn(2+) stimulation of the Dopa/tyrosine-sulfating activity of SULT1A3. A model is proposed, with reference to the reported x-ray crystal structure of SULT1A3, to explain how the normal role of SULT1A3 in dopamine regulation may be subverted in the presence of Mn(2+). These studies could be relevant in understanding the stereoselective action of SULT1A3 on chiral drugs.