Project Title

Vitamin D and its in vitro therapeutic action mediated through VDR rather than PDIA3

Authors' Affiliations

Jaeden Pyburn, Department of Health Sciences, College of Public Health, East Tennessee State University, Johnson City, TN. Theodore Hagg, Department of Biomedical Science, Quillen College of Medicine, East Tennessee State University, Johnson City, TN. Matthew Keasey, Department of Biomedical Science, Quillen College of Medicine, East Tennessee State University, Johnson City, TN.

Location

Culp Room 217

Start Date

4-6-2022 9:45 AM

End Date

4-6-2022 10:00 AM

Faculty Sponsor’s Department

Biomedical Sciences

Name of Project's Faculty Sponsor

Matthew Keasey

Additional Sponsors

Dr. Cuihong Jia, Dr. Sean Fox

Classification of First Author

Undergraduate Student

Competition Type

Non-Competitive

Type

Boland Symposium

Project's Category

Cell Biology

Abstract or Artist's Statement

Brain calcification is a common occurrence in the aging process, with over 20% of individuals over the age of 65 showing hardened plaques in the basal ganglia. Loss of the vitamin D receptor (VDR) in transgenic mice leads to formation of calcified plaques in the basal ganglia and thalamus within the mice. Vitamin D signals through two known vitamin D responsive proteins, protein disulfide isomerase A3 (PDIA3) and VDR. In vitro, vitamin D has been demonstrated to suppress calcification in osteoblast-like cells. Here, we aim to elucidate which of PDIA3 or VDR transduce vitamin D mediated suppression of calcification in vitro. PDIA3 or VDR were selectively knocked out in human osteosarcoma (SaOs) cells using CRISPR/CAS9 technology to generate PDIA3 -/- or VDR -/- cells. Knockout for PDIA3 or VDR was confirmed by RT-qPCR assay or western blot analysis. The calcification of SaOs-2 cells was induced with treatment of β-glycerophosphate along with ascorbic acid allowing for determination of whether loss of PDIA3 or VDR would lead to altered calcium deposition. Cells null for PDIA3 but not VDR grew at a significantly slower rate than wild-type (WT) cells. Intriguingly, PDIA3 and VDR -/- cells displayed significantly more calcification relative to WT control cells. Calcitriol or the synthetic analogue EB-1089 suppressed calcification in vitro in WT and PDIA3 -/- but not VDR -/- cells as measured by alizarin red staining. These data suggest VDR is critical for mediating vitamin D’s inhibition of calcification in vitro, and that PDIA3 has a role in suppressing calcification. This study provides novel insights into vitamin D signaling and provides a foundation for further study and understanding of vitamin D related pathologies.

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Apr 6th, 9:45 AM Apr 6th, 10:00 AM

Vitamin D and its in vitro therapeutic action mediated through VDR rather than PDIA3

Culp Room 217

Brain calcification is a common occurrence in the aging process, with over 20% of individuals over the age of 65 showing hardened plaques in the basal ganglia. Loss of the vitamin D receptor (VDR) in transgenic mice leads to formation of calcified plaques in the basal ganglia and thalamus within the mice. Vitamin D signals through two known vitamin D responsive proteins, protein disulfide isomerase A3 (PDIA3) and VDR. In vitro, vitamin D has been demonstrated to suppress calcification in osteoblast-like cells. Here, we aim to elucidate which of PDIA3 or VDR transduce vitamin D mediated suppression of calcification in vitro. PDIA3 or VDR were selectively knocked out in human osteosarcoma (SaOs) cells using CRISPR/CAS9 technology to generate PDIA3 -/- or VDR -/- cells. Knockout for PDIA3 or VDR was confirmed by RT-qPCR assay or western blot analysis. The calcification of SaOs-2 cells was induced with treatment of β-glycerophosphate along with ascorbic acid allowing for determination of whether loss of PDIA3 or VDR would lead to altered calcium deposition. Cells null for PDIA3 but not VDR grew at a significantly slower rate than wild-type (WT) cells. Intriguingly, PDIA3 and VDR -/- cells displayed significantly more calcification relative to WT control cells. Calcitriol or the synthetic analogue EB-1089 suppressed calcification in vitro in WT and PDIA3 -/- but not VDR -/- cells as measured by alizarin red staining. These data suggest VDR is critical for mediating vitamin D’s inhibition of calcification in vitro, and that PDIA3 has a role in suppressing calcification. This study provides novel insights into vitamin D signaling and provides a foundation for further study and understanding of vitamin D related pathologies.