Bone tissue specific delivery of dietary metabolite phenolic acids using Clostridial collagenase collagen binding domain

Authors: CAVINESS, PERRY - Arkansas Children'S Nutrition Research Center (ACNC); LAZARENKO, OXANA - Arkansas Children'S Nutrition Research Center (ACNC); BLACKBURN, MICHAEL - Arkansas Children'S Nutrition Research Center (ACNC); CHEN, JIN-RAN - Arkansas Children'S Nutrition Research Center (ACNC)

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 3/11/2024
Publication Date: N/A
Citation: N/A

Interpretive Summary: Dietary metabolites of phenolic acids, hippuric acid (HA) and 3-(3-hydroxyphenyl) propionic acid (3-3-PPA), suppress bone resorption and show potential as novel anti-bone resorption therapeutics. To avoid potential degradation, HA and 3-3-PPA were cross-linked to Clostridial collagenase collagen binding domain (CBD) and the ability of cross-linked HA or 3-3-PPA (HA-CBD or 3-3-PPA-CBD) to specifically target bone was investigated in mouse models. In addition, HA-CBD/3-3-PPA-CBD activities on bone cells were determined and HA or 3-3-PPA epigenetic effects on bone were investigated.

Technical Abstract: Objectives Dietary metabolites of phenolic acids, hippuric acid (HA) and 3-(3-hydroxyphenyl) propionic acid (3-3-PPA), suppress bone resorption and show potential as novel anti-bone resorption therapeutics. To avoid potential degradation, HA and 3-3-PPA were cross-linked to Clostridial collagenase collagen binding domain (CBD) and the ability of cross-linked HA or 3-3-PPA (HA-CBD or 3-3-PPA-CBD) to specifically target bone was investigated in mouse models. In addition, HA-CBD/3-3-PPA-CBD activities on bone cells were determined and HA or 3-3-PPA epigenetic effects on bone were investigated. Methods HA or 3-3-PPA were crosslinked to CBD using EDC/NHS crosslinker and modification was confirmed with proteomics. Additionally prior to injection into pregnant mouse model, N-terminal Cys of HA-CBD/3-3-PPA-CBD was conjugated with Alexa Fluor 488 dye. After 4 hr., mice were sacrificed, bone tissue was collected from both dams and fetus, decalcified using EDTA, prepared for histology and observed under an epifluorescent microscope. Results HA-CBD/3-3-PPA CBD were shown to be as effective at suppressing osteoclastogenesis as unconjugated HA/3-3-PPA. In pregnant mice, HA-CBD/3-3-PPA-CBD localized in dam tibia and fetal calvaria. Bone marrow cells (BMC) and bone marrow plasma (BMP) from dams showed an increase in HA following HA-CBD injection (BMC, Control: 1.76 µmole HA/µg cell lysate, HA-CBD: 2.16 µmole HA/µg cell lysate; BMP, Control: 0.68 µmole/µg cell lysate, HA-CBD: 1.36 µmole/µg cell lysate). Osteoclast promoting genes, MMP9 and Cav1, were suppressed in Raw 264.7 cells following HA-CBD/3-3-PPA-CBD treatment. Investigation into HA/3-3-PPA mechanism, revealed that DNA methylation (%5-mC) is decreased following HA or 3-3-PPA treatment and %5-mC levels have a strong negative correlation with bone volume (R = -0.506). In addition, NADPH levels in Raw 264.7 cells were suppressed following HA/3-3-PPA treatment. Conclusions In pregnant mice, HA-CBD/3-3-PPA-CBD localized to bone tissue in both dam and fetus, showing an ability to cross the placental barrier and impact maternal and fetal bone tissue. In addition, HA/3-3-PPA induced decrease in %5-mC was negatively correlated with bone volume, suggesting these dietary metabolites epigenetically regulated bone development. Funding Sources This study is supported by funds from the USDA-ARS and NIH.