# Vesicle Trafficking and Osteoblast Function

> **NIH NIH R21** · STANFORD UNIVERSITY · 2023 · $171,246

## Abstract

Project Summary
Osteoporosis is a disease of skeletal fragility that causes fractures in 50% of women and 25% of men over age
50. The most commonly prescribed anti-resorptive osteoporosis medications cannot cure osteoporosis, while
currently available bone-building anabolic osteoporosis medications are limited by waning efficacy. There is still
a great unmet need for safe and sustained approaches to increasing bone formation. We have shown
that 2.3-kb rat type I collagen promoter-driven green fluorescent protein (Col2.3GFP) is highly expressed in
mature osteoblasts. We isolated Col2.3GFP(hi) osteoblasts from bones, cultured bone chips, and directed
differentiation of embryonic stem cells, and by RNA-sequencing identified 593 genes that are enriched in mature
osteoblasts. Gene ontology (GO) analysis identified ER-to-Golgi vesicle trafficking as the most highly enriched
GO term. Our preliminary data reveal that transient knockdown in MC3T3 osteoblasts of several vesicle
trafficking genes results in increased mineralized nodule formation and accelerated osteoblast marker
expression. Our central hypothesis is that disruption of vesicle trafficking impairs bone formation due to
collagen overmodification and hypermineralization. We will leverage several innovative methods: direct
reprogramming of fibroblasts to derive induced osteoblasts, CRISPR/Cas9 gene editing to delete vesicle
trafficking genes in induced osteoblasts, Col2.3GFP as a cell-autonomous osteoblast reporter, and
subcutaneous transplantation of gene-edited osteoblasts to assess in vivo bone formation. We screened vesicle
trafficking genes with transient siRNA knockdown, and selected 9 genes (Preb, Stx5a, Rab2a, Gosr2, Bet1,
Ramp1, Arf4, Cog6, Pacs1) whose knockdown increased mineralized nodule formation, osteoblast marker
expression, and ER stress. In Aim 1 we will determine whether disruption of vesicle trafficking increases
mineralization due to collagen overmodification. We will perform permanent knockdown by CRISPR/Cas9 gene
editing of each gene in mouse and human induced osteoblasts with the Col2.3GFP osteoblast reporter and
assess osteoblast marker expression and mineralized nodule formation. We will measure collagen production
and post-translational modification by prolyl hydroxylation, and determine whether inhibition of collagen
overmodification can restore osteoblast function. In Aim 2 we will determine whether disruption of vesicle
trafficking impairs bone formation in vivo. We will examine bone formation in vivo by subcutaneous
transplantation of gene-edited mouse and human iOBs. We will determine whether inhibition of collagen
overmodification restores bone formation in vivo. Successful completion of these aims will identify novel genes
involved in osteogenesis as potential therapeutic targets for the treatment of osteoporosis, and will provide
mechanistic insights into the role of vesicle trafficking machinery in osteoblast function.

## Key facts

- **NIH application ID:** 10709486
- **Project number:** 5R21AR081073-02
- **Recipient organization:** STANFORD UNIVERSITY
- **Principal Investigator:** JOY Y WU
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $171,246
- **Award type:** 5
- **Project period:** 2022-09-23 → 2024-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10709486

## Citation

> US National Institutes of Health, RePORTER application 10709486, Vesicle Trafficking and Osteoblast Function (5R21AR081073-02). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10709486. Licensed CC0.

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