Defense Date

6-10-2021

Graduation Date

Summer 8-7-2021

Availability

One-year Embargo

Submission Type

dissertation

Degree Name

PhD

Department

Pharmacology-Toxicology

School

School of Pharmacy

Committee Chair

Paula A. Witt-Enderby

Committee Member

David A. Johnson

Committee Member

Rehana K. Leak

Committee Member

Jane Cavanaugh

Committee Member

Deborah L. Galson

Keywords

Melatonin, Bone, MEK1/2, MEK5, Osteoblasts, Osteoclasts, bone formation, bone microarchitecture, bone biomechanics

Abstract

Melatonin, the main endogenous hormone to entrain the circadian system, is not limited to its role in regulating the sleep-wake cycle; rather, it affects a wide variety of systems involving antioxidant, anti-inflammation, blood pressure regulation, seasonal reproduction, ovarian physiology, and immune function. Driven by the diversity of its action, melatonin for a long time has also been studied in the field of bone and mineral research both clinically and pre-clinically. Exogenous administration of melatonin in clinical trials in perimenopausal women (MOPS; NCT01152580); or postmenopausal women with osteopenia (MelaOST; NCT01690000 and MOTS; NCT01870115) confirmed improvement of bone mineral density (BMD) (MelaOST; MOTS) and bone marker turnover status (MOPS, MOTS). Studies involving preclinical animal models also revealed melatonin’s effect in improving age-related bone loss and BMD with efficacy similar to a therapeutically relevant estrogen and progesterone hormone therapy. At the cellular level, this increase in BMD by melatonin was accompanied by increases in the levels of osteogenic proteins, pErk1/2, and pErk5, indicating the potential role of the MAPKs, MEK1/2/ERK1/2 and MEK5/ERK5 pathways in mediating melatonin’s action. To investigate this further, the goal of this project was to study the role of MEK1/2 and MEK5 in regulating melatonin-mediated osteoblast and osteoclast differentiation and function in vitro and in vivo and their role in modulating bone density, quality, strength, and formation. In vitro, using small-molecule inhibitors and a co-culture model of human bone marrow-derived mesenchymal stem cells (hMSCs) and peripheral blood monocytes (hPBMCs), it was discovered that melatonin’s stimulating effect on osteoblastogenesis is mediated through MT2 melatonin receptors, MEK1/2, MEK5, and perhaps PPARγ and GLUT4. To further confirm the involvement of MEK1/2 and MEK5 in melatonin’s effect, CRISPR/Cas9 knockout approaches were used to generate MEK1KO or MEK5KO hMSCs and mouse mesenchymal stem cells (mMSCs). Both monoculture and co-culture models were developed using these MEK1KO or MEK5KO MSCs with a goal to study the role of melatonin, melatonin receptors, MEK1/2, and MEK5 in osteoblast and osteoclast differentiation and communication between these cells. In both human and mouse MSCs, melatonin’s effect on osteoblastogenesis was occurring strictly through MT2R-mediated actions on MEK5 and/or MEK1 and not through an indirect action of melatonin on MEK5 or MEK1, consistent with the findings using small molecule inhibitors. In vivo and using small molecule inhibitors or a CRISPR/Cas9 knockout approach, it was further determined that Mek1/2 and 5 were primary drivers underlying melatonin’s actions on bone formation, bone microarchitecture, and bone biomechanics. In the small molecule inhibitor study where Balb(c) mice (female) were injected with melatonin in the absence or presence of selective MEK1/2, MEK5, or MEK1/2/5 inhibitors for 45 days, it was demonstrated that melatonin, through MEK1/2 and MEK5, increased osteogenic protein expression (Runx2, Bmp-2, Fra-1, Opg) and decreased metabolic protein, Pparγ expression; and also modulated bone microarchitecture (i.e., trabecular number, separation, and connectivity density) and bone mechanical properties (i.e., ultimate stress). A mouse calvarial defect model was developed using PLGA scaffolds seeded with mMSCs (wildtype, control, Mek1KO, Mek5KO) and placed into critical size calvarial defects created in Balb(c) mice (male and female) followed by treatment with vehicle or melatonin nightly for 90 days. This study demonstrated the involvement of Mek1 and Mek5 in new bone formation induced by melatonin in both genders supporting the findings in vitro in human and mouse MSCs. Gender-specific analyses of the calvarial data revealed unique gender differences in melatonin’s effect and kinase interaction with melatonin. These mechanisms of action demonstrating a unique role for Mek1/2 and Mek5 in mediating both osteogenic and metabolic pathways as well as demonstrating specificity of action in a gender-specific manner opens up new avenues of research examining conditions known to promote bone loss (i.e., diabetes, aging) in males and females and novel therapeutic strategies and agents to modulate bone loss to prevent fracture and mortality.

Language

English

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