Defense Date

3-3-2006

Graduation Date

Spring 2006

Availability

Immediate Access

Submission Type

dissertation

Degree Name

PhD

Department

Pharmacology-Toxicology

School

School of Pharmacy

Committee Chair

Paula Witt-Enderby

Committee Member

David A. Johnson

Committee Member

Wilson S. Meng

Committee Member

Kyle W. Selcer

Keywords

Differentiation, ERK, MAP-K, MEK, Melatonin, Osteoblasts

Abstract

Current osteoporosis therapy is directed toward inhibiting the activity of bone-resorbing osteoclasts. Decreased bone producing osteoblast efficiency however, is largely responsible for osteoporosis development. Therefore, novel osteoporosis treatments should stimulate osteoblast formation and activity. Accordingly, the purpose of this study was to determine melatonin's role and subsequent mechanism(s) involved in differentiating human adult mesenchymal stem cells (hAMSCs) into osteoblasts as assessed by alkaline phosphatase activity (ALP) and determine whether receptor compartmentalization facilitates these signaling mechanisms. Melatonin significantly increased osteogenic medium-induced alkaline phosphatase activity, an enzyme associated with functional osteoblasts, indicating melatonin enhances osteoblast differentiation. The mitogen activated protein kinase signaling mechanisms has been implicated in osteoblast differentiation. Based on these findings, we hypothesized that melatonin acts through the mitogen activated protein kinase signaling cascade to enhance hAMSC differentiation into an osteoblast as assessed by alkaline phosphatase activity. Acute melatonin exposure to hAMSC stimulated extracellular regulated kinase (ERK) phosphorylation without affecting total ERK protein levels. Conversely, co-incubation with melatonin and the MEK inhibitor PD98059 prevented the melatonin-mediated enhancement in ALP activity in hAMSCs. Taken together, these findings indicate melatonin enhances ALP activity via activation of the mitogen activated protein kinase (MAP-K) cascade. Protein Kinase A (PKA), epidermal growth factor receptor (EGFR), and matrix metalloproteinase (MMP) activation were evaluated as potential upstream inputs of the MAP-K activation. Subsequent co-incubation of melatonin with specific PKA, EGFR, and MMP inhibitors revealed that EGFR and possibly MMP but not PKA activate the MAP-K cascade in these cells. The potential for compartmentalization of the melatonin receptor and associative signaling pathways was assessed by immunoprecipitation analysis. The MT2 receptor was immunprecipitated in the presence of vehicle, melatonin or melatonin + luzindole and the pellet was subjected to western blot analysis using antibodies against Gi, EGFR, beta arrestin 1, 2, MEK and ERK (1/2). Stem cells exposed to melatonin showed that MT2 receptors do complex with Giα2, beta arrestin, and MEK in an agonist-dependent manner. These findings indicate in differentiating hAMSCs exposed chronically to melatonin, beta-arrestin scaffolds form with MT2 receptors bringing together a signaling chain that may facilitate activation of the MAP-K cascade and hence, their differentiation into osteoblasts.

Format

PDF

Language

English

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