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Clinkenbeard, E. L.

Publications and source records attributed to Clinkenbeard, E. L..

2 recordsLinked to original sources
bioRxiv2021

Suppression of sost/sclerostin and dikkopf-1 promote intervertebral disc structure in mice

Intervertebral disc (IVD) degeneration is a leading cause of low back pain and characterized by accelerated extracellular matrix breakdown and IVD height loss but there is no approved pharmacological therapeutic. Deletion of Wnt signaling receptor Lrp5 induces IVD degeneration and suggests that Wnt signaling in the IVD may be responsive to inhibition of Wnt signaling inhibitors sost(gene)/sclerostin(protein) or dickkopf-1 (dkk1). Anti-sclerostin antibody (Scl-Ab) is an FDA-approved bone therapeutic that activates Wnt signaling. We (1) determined if pharmacological neutralization of sclerostin, dkk1 or their combination stimulate Wnt signaling and promote IVD structure and (2) determined the extent of the response of the IVD to global, persistent deletion of sost. Nine-week-old C57Bl/6J female mice (n=6-7/grp) were subcutaneously injected 2x/wk for 5.5 wk with scl-Ab (25 mg/kg), dkk1-Ab (25 mg/kg), 3:1 scl-Ab/dkk1-Ab (18.75:6.25 mg/kg) or vehicle (Veh). Separately, IVD of sost KO and WT (wildtype) mice (n=8, grp) were harvested at 16 weeks of age. First, compared to vehicle, scl-Ab, dkk1-Ab and 3:1 scl-Ab/dkk1-Ab similarly increased lumbar IVD height and {beta}-catenin gene expression. Despite these similarities, scl-Ab decreased cellular stress-related heat shock protein gene expressions while neither dkk1-Ab nor scl-Ab/dkk1-Ab altered the same. Genetically and compared to WT, sost KO increased MRI-determined hydration and proteoglycan staining in the IVD. Notably, persistent deletion of sost was compensated by upregulation of dkk1, which consequently reduced the cell nuclear fraction for Wnt signaling transcription factor {beta}-catenin in whole IVD. Lastly, RNA-sequencing pathway analysis confirmed the compensatory suppression of Wnt signaling and determined a reduction of cellular stress pathways. Together, suppression of sost/sclerostin or dkk1 each promote IVD structure by stimulating Wnt signaling, but sclerostin and dkk1 may differentially regulate cellular stress pathways. Ultimately, postmenopausal women prescribed scl-Ab injections to prevent vertebral fracture may also benefit from a restoration of IVD height and health. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=72 SRC="FIGDIR/small/449486v2_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@db2e17org.highwire.dtl.DTLVardef@1af1bbborg.highwire.dtl.DTLVardef@145cc4corg.highwire.dtl.DTLVardef@167a19e_HPS_FORMAT_FIGEXP M_FIG C_FIG Suppression of Wnt signaling inhibitors by genetic or pharmacological approaches promotes intervertebral disc structure and hydration by Wnt signaling. However, persistent activation of Wnt signaling induces a compensatory reduction of Wnt signaling that shifts IVD cells toward a chondrocyte-like (CLC) phenotype. AF: annulus fibrosus, NC: notochordal cell, NP: nucleus pulposus, PG: proteoglycan

bioengineering↗
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↗