# Mechanisms of Physiologic and Pathologic Osteoclastogenesis

> **NIH NIH R01** · WASHINGTON UNIVERSITY · 2020 · $335,500

## Abstract

ABSTRACT:
The transcription factor NF-kB is expressed ubiquitously in all cell types and is readily activated by numerous
factors and cytokines. Baseline NF-kB activity is essential for skeletal development and physiologic cellular
functions. In contrast, its exacerbated and often uncontrolled activity during inflammation leads to undesired
harmful effects with major dysfunctional consequences including osteolysis. Hence, therapies targeting NF-kB
have been highly pursued to combat most inflammatory diseases. Unfortunately, most available therapies are
inefficient owing to lack of selectivity in such complex and ubiquitous signaling pathway wherein the essential
beneficial functions of NF-kB are blocked along side the harmful effects leading to detrimental outcomes.
Therefore, there is an unmet need to decode NF-kB signaling to identify specific targets that assign
signal specificity and distinguish between physiologic and pathologic functions. To address this critical
knowledge gap, we focused on RANKL-induced osteoclastogenesis as a proof of concept and set out to
decipher the NF-kB molecular machinery and identify the signal-specific molecular signature that controls this
response in osteoclast progenitors and maintains skeletal homeostasis. We hypothesize that the IKK scaffold
IKKγ/NEMO serves as a platform that site-specifically assembles unique signal activating or suppressing
protein complexes in cell and stimulus specific manners. This hypothesis is based on recent advances
implicating NEMO as a scaffold that integrates signaling molecules in response to a wide range of stimuli at
lysine (K) specific sites (refer to Fig 2). These modifications include, lysine poly-ubiquitination,
SUMOylation, and according to our novel finding, ISGylation; a process of attaching the ubiquitin-like protein,
ISG15 (IFN-stimulated gene) to target proteins. We conduced comprehensive NEMO lysine mutational
analysis and identified the NEMO K270 residue as a crucial RANKL-regulation target. Specifically, NEMO
harboring K270A mutation (NEMOK270A) elicits exacerbated osteoclastogenesis. More importantly, myeloid
knock-in mice of the NEMOK270A that we generated displayed severe osteopenia and osteolysis.
Mechanistically, autophagy is significantly decreased in NEMOK270A BMMs. Furthermore, proteomic screen
identified interferon-stimulated gene-15 (ISG15) as a potential regulator of osteoclastogenesis and autophagy.
Thus, our overarching hypothesis is: RANKL-induced binding of ISG15 to NEMO at K270 is essential to
restrain osteoclastogenesis by assembling a negative-feedback response. We further posit that mutating K270
hinders this regulatory process leading to reduced autophagy and uncontrolled osteoclastogenesis. Our aims
are: Aim 1: Determine the mechanism by which NEMO, through its K270 site, maintains physiologic
and restrains pathologic/exacerbated osteoclastogenesis.
Aim 2: Determine the role of RANKL-induced ISG15 as the ubiquitin-like protein that facilitat...

## Key facts

- **NIH application ID:** 9889901
- **Project number:** 5R01AR072623-03
- **Recipient organization:** WASHINGTON UNIVERSITY
- **Principal Investigator:** YOUSEF ABU-AMER
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $335,500
- **Award type:** 5
- **Project period:** 2018-04-01 → 2023-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9889901

## Citation

> US National Institutes of Health, RePORTER application 9889901, Mechanisms of Physiologic and Pathologic Osteoclastogenesis (5R01AR072623-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9889901. Licensed CC0.

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