Biology subjects

Noonan, M. L.

Publications and source records attributed to Noonan, M. L..

3 recordsLinked to original sources

bioRxiv2024

Dynamic Single Cell Transcriptomics Defines Kidney FGF23/KL Bioactivity and Novel Segment-Specific Inflammatory Targets

FGF23 via its coreceptor Klotho (KL) provides critical control of phosphate metabolism, which is altered in rare and very common syndromes, however the spatial-temporal mechanisms dictating renal FGF23 functions remain poorly understood. Thus, developing approaches to modify specific FGF23-dictated pathways has proven problematic. Herein, wild type mice were injected with rFGF23 for 1, 4 and 12h and renal FGF23 bioactivity was determined at single cell resolution. Computational analysis identified distinct epithelial, endothelial, stromal, and immune cell clusters, with differential expressional analysis uniquely tracking FGF23 bioactivity at each time point. FGF23 actions were sex independent but critically relied upon constitutive KL expression mapped within proximal tubule (S1-S3) and distal tubule (DCT/CNT) cell sub-populations. Temporal KL-dependent FGF23 responses drove unique and transient cellular identities, including genes in key MAPK- and vitamin D-metabolic pathways via early- (AP-1-related) and late-phase (EIF2 signaling) transcriptional regulons. Combining ATACseq/RNAseq data from a cell line stably expressing KL with the in vivo scRNAseq pinpointed genomic accessibility changes in MAPK-dependent genes, including the identification of FGF23-dependent EGR1 distal enhancers. Finally, we isolated unexpected crosstalk between FGF23-mediated MAPK signaling and pro-inflammatory TNF receptor activation via NF-{kappa}B, which blocked FGF23 bioactivity in vitro and in vivo. Collectively, our findings have uncovered novel pathways at the single cell level that likely influence FGF23-dependent disease mechanisms. Translational statementInflammation and elevated FGF23 in chronic kidney disease (CKD) are both associated with poor patient outcomes and mortality. However, the links between these manifestations and the effects of inflammation on FGF23-mediated mineral metabolism within specific nephron segments remain unclear. Herein, we isolated an inflammatory pathway driven by TNF/NF-{kappa}B associated with regulating FGF23 bioactivity. The findings from this study could be important in designing future therapeutic approaches for chronic mineral diseases, including potential combination therapies or early intervention strategies. We also suggest that further studies could explore these pathways at the single cell level in CKD models, as well as test translation of our findings to interactions of chronic inflammation and elevated FGF23 in human CKD kidney datasets.

physiology↗

bioRxiv2022

Gabapentin Disrupts Binding of Perlecan to the α2δ1 Voltage Sensitive Calcium Channel Subunit and Impairs Skeletal Mechanosensation

Our understanding of how osteocytes, the principal mechanosensors within bone, sense and perceive force remains unclear. Previous work identified "tethering elements" (TEs) spanning the pericellular space of osteocytes and transmitting mechanical information into biochemical signals. While we identified the heparan sulfate proteoglycan perlecan (PLN) as a component of these TEs, PLN must attach to the cell surface to induce biochemical responses. As voltage-sensitive calcium channels (VSCCs) are critical for bone mechanotransduction, we hypothesized that PLN binds the extracellular 2{delta}1 subunit of VSCCs to couple the bone matrix to the osteocyte membrane. Here, we showed co-localization of PLN and 2{delta}1 along osteocyte dendritic processes. Additionally, we quantified the molecular interactions between 2{delta}1 and PLN domains and demonstrated for the first time that 2{delta}1 strongly associates with PLN via its domain III. Furthermore, 2{delta}1 is the binding site for the commonly used pain drug, gabapentin (GBP), which is associated with adverse skeletal effects when used chronically. We found that GBP disrupts PLN::2{delta}1 binding in vitro, and GBP treatment in vivo results in impaired bone mechanosensation. Our work identified a novel mechanosensory complex within osteocytes composed of PLN and 2{delta}1, necessary for bone force transmission and sensitive to the drug GBP. This work provides insights into the mechanisms underlying mechanotransduction and will inform future studies to understand the mechanisms responsible for the negative effects of GBP on bone.

molecular biology↗

bioRxiv2020

Generation of two Multipotent Mesenchymal Progenitor Cell Lines Capable of Osteogenic, Mature Osteocyte, Adipogenic, and Chondrogenic Differentiation

Differentiation of multi-potent mesenchymal progenitor cells give rise to several tissue types including bone, cartilage, and adipose. In addition to the complication arising from the numerous spatial, temporal, and hormonal factors that regulate lineage allocation, targeting of these cells in vivo is challenging, making mesenchymal progenitor cell lines valuable tools to study both tissue development and the differentiated cell types. Mesenchymal stem cells (MSCs) can be isolated from humans and animals; however, obtaining homogenous, responsive cells in a reproducible fashion can be problematic. As such, we have developed two novel mesenchymal progenitor cell (MPC) lines, MPC1 and MPC2, which were generated from the bone marrow of male C57BL/6 mice. These cells were immortalized using the temperature sensitive large T-antigen, allowing for thermal control of proliferation and differentiation. Both MPC1 and MPC2 cell lines are capable of osteogenic, adipogenic, and chondrogenic differentiation. Under osteogenic conditions both cell lines formed discrete mineralized nodules, staining for alizarin red and alkaline phosphatase, while expressing high levels of osteogenic genes including Sost, Fgf23, and Dmp1. Sost and Dmp1 mRNA levels were drastically reduced with parathyroid hormone, thus recapitulating in vivo responses. MPC cells secreted both the intact (iFGF23) and C-terminal (cFGF23) forms of endocrine hormone FGF23, which was upregulated in the presence of 1,25 dihydroxy vitamin D (1,25D). In addition to osteogenic differentiation, both cell lines also rapidly entered the adipogenic lineage, expressing several adipose markers after only 4 days in adipogenic media. MPC cells were also capable of chondrogenic differentiation, displaying increased expression of common cartilage genes including aggrecan, sox9, and cartilage oligomeric matrix protein. With the ability to differentiate into multiple mesenchymal lineages and mimic in vivo responses of key regulatory genes/proteins, MPC cells are a valuable model to study factors that regulate mesenchymal lineage allocation as well as the mechanisms that dictate transcription, protein modification, and secretion of these factors.

cell biology↗