The focus of the research in my laboratory is on the signaling mechanisms underlying melatonin receptor function and regulation.  I use biochemical, pharmacological and molecular biological approaches to assess the signaling pathways utilized by melatonin receptors to produce a cellular response.  I study these responses using both in vitro and in vivo model systems. In addition, I am involved with drug discovery whereby I work in collaboration with medicinal chemists to develop novel, subtype selective melatonin receptor ligands or therapeutic agents for disease management or treatment. In recent years, I have moved my studies in cells to pre-clinical and clinical models. Along with my collaborators, we are assessing melatonin effects on bone remodeling in rodents as well as in perimenopausal women (please see link to clinical trial flyer, Tribune Review news article, and information)
 
CELLULAR DIFFERENTIATION PATHWAYS
In recent years, the primary focus of my research has been on cellular differentiation (For complete papers, see my cv and papers Witt-Enderby et al., 2000; Bordt et al., 2001; Witt-Enderby et al., 2003; Jarzynka et al., 2006; Bondi et al., 2008).  In particular, my lab has shown that chronic melatonin exposure can produce a hyper-elongation of cells that is driven by changes in microtubule dynamics.  We have shown that melatonin drives these changes in cellular morphology through coordinated scaffolding and internalization of “desensitized” melatonin receptors with Gi proteins, beta arrestins, MEK 1/2 and ERK 1/2.  Additionally, epidermal growth factor (EGF) receptor activation is essential.  We show that melatonin receptors can transactivate EGF receptors through matrix metalloproteinases.  Matrix metalloproteinase activation, then, can lead to the liberation of heparin-bound EGF to allow for EGF to act as an autocrine factor on its own receptor.  See Figure 1

APPLICATION TO BONE STEM CELL DIFFERENTIATION INTO OSTEOBLASTS
We have been able to generalize this signaling pathway from recombinant cell models to human adult mesenchymal stem cell models.  That is, we have found a common signaling pathway used by either the MT1 or MT2 melatonin receptor to enhance cellular differentiation whether the endpoint is changes in cellular morphology or bone stem cell differentiation into an osteoblast (Radio et al., 2006).  My most recent work focuses on assessing melatonin effects on bone density, bone regeneration and in the prevention of osteoporosis. In effect, we have moved our work in cells to pre-clinical and clinical models. Please see adevrtisement for clinical trial: clinical trial flyer and information. If you are interested and eligible, please give us a call. We have carried this idea further to mammary tissue.  Here, we show that melatonin, given in the drinking water of HER2/neu female mice, enhances the extent of ductal differentiation.  Highly differentiated ductules in the mammary tissue have been associated with a reduction in breast cancer risk in humans. (See reviews Grant et al., 2009; Witt-Enderby et al., 2006; and http://pittsburghlive.com/x/pittsburghtrib/news/cityregion/s_506093.html).

APPLICATION OF MELATONIN AND EFFECTS ON MICROTUBULES TO ALTERED SLEEP/WAKE PATTERNS IN THOSE AFFLICTED WITH Alzheimer’s disease
Another area of my research focuses on the relationship between melatonin receptors, G-proteins and microtubules.  Previously, we showed that chronic melatonin exposure produced microtubule rearrangements in CHO cells expressing the human MT1 melatonin receptor.  In the same time frame, MT1 receptor desensitization occurred as well as an increase in Giα.  An increase in the “heterotrimeric” Giα–GDP state of the G-protein would prevent a transfer of signal from the Gi protein to adenylyl cyclase resulting in a “supersensitization” of adenylyl cyclase, which is what we, too, observed. 
We explored further this relationship between MT1 melatonin receptor desensitization and microtubule polymerization by disrupting microtubule polymerization using microtubule-disrupting agents like colchicine and demecolcine.  Here, we showed that when microtubules were prevented from polymerizing during the melatonin exposure that the MT1 melatonin receptor remained sensitive.  In addition, by using multiple approaches, we showed that an increase in the dissociated Giα-GTP state of the G protein occurred.  This increase in Giα-GTP would increase the transfer of signal from the melatonin receptor to adenylyl cyclase, which is what we, too, observed.  The downstream effects of this microtubule disruption were also explored.  Our lab has shown that microtubule effects through Gi enhanced protein kinase C (PKC) activity.  PKC has been shown to be involved in mediating the circadian entraining effects of melatonin in the master biological clock (Jarzynka et al., 2006). 
Sleep/wake disturbances are commonly seen in patients with Alzheimer’s disease (AD).  In addition, people with AD have disrupted circadian timekeeping systems, disrupted microtubules and alterations in melatonin receptor levels in their brains, including the master biological clock.  The data from our lab suggest that melatonin may be useful as a therapeutic to treat the altered sleep/wake disturbances in people afflicted with AD (See Jarzynka et al., 2009).

USE OF MELATONIN AS AN ADJUVANT TO HORMONE REPLACEMENT THERAPY (HRT) TO REDUCE BREAST CANCER RISK ASSOCIATED WITH HRT
The goal of my research is to apply the knowledge gained from the bench to the bedside whereby novel therapeutic approaches using melatonin for disease management or prevention can be investigated.  In a recently published review (Witt-Enderby et al., 2006), the use of melatonin in the prevention of osteoporosis, cancer and as an adjuvant therapy was discussed.  Also, my lab was funded by the Susan G. Komen Breast Cancer Foundation to study the use of melatonin as an adjuvant to hormone replacement therapy (HRT) as a means of reducing the breast cancer risk associated with HRT using an in vivo model of HER2/neu breast cancer.  Studies are currently underway exploring this exciting new area of research.

The development of my research program was the result of a lot of hard work from my past and present graduate students.  My sincere gratitude goes out to my past students:

Renee MacKenzie, M.S.  (Masters student, 1996-1997)
Marla (Jones) Kress, M.S. (Masters student, 1996-1998)
Joerg Brockmann, Ph.D. (B.S. Pharmacy intern in the spring and fall, 1997)
Emily Weiss, ? (Summer undergraduate intern from the Pittsburgh Tissue Engineering Institute, summer 1997)
Megan (Gillen) Marks, Ph.D. (Rotation student from 1996-1997)
Ann Gorman, B.S.,  Pharm.D. (B.S. and Pharm.D. student of mine from 1998-2000)
Jennifer Bennett, M.S. (Biology graduate student, 2000-2001)
Raelene McKeon, M.S. (Biology graduate student, 1998-2000)
Erin Litten, M.S. (Biology graduate student, 1998-2000)
Tejal Parekh, M.S. (Medicinal Chemistry graduate student, 1997-2000)
B. Justin Krawitt, (M.S. student in Pharmacology and Toxicology, 1998-2002)
Shannon Bordt, P.A. (Summer intern, 1999-2000)
Paul Ignatius, M.D. (Post-baccalaureate student, 2000-2001)
Michael Jarzynka, Ph.D. (Doctoral student, 1997-2003)
Deepshikha K. Passey, M.S. (Masters student, 2000-2003)
Corry Bondi, M.S. (Masters student, 2001-2006; Doctoral student 2008-present)
Nicholas Radio, Ph.D. (Doctoral student, 2001-2006)
Nagarjun Konduru, (Masters student, 2003-2005)
Shalini Sethi (Doctoral student, 2004-present)
Bill Clafshenkel, (Masters student, 2006-2009; Doctoral student 2009-present)
Justin Julius, (Pharm. D. student, 2006-2008)
Tracy King, Pharm. D. (Fellow, 2006-2008)
Bala Sunder Reddy, (Doctoral student, 2007-present)
Mary Kotlarczyk (Doctoral student, 2008-present)