Molecular and Cellular Physiology Laboratory

Ramaley C373

Department of Integrative Physiology
University of Colorado
Boulder, CO 80309-0354
phone: 303-735-6712
fax: 303-492-0811

Research Interests

The primary research focus of the Molecular and Cellular Physiology Laboratory is on understanding the molecular mechanisms underlying changes in gene expression during adaptation of muscle and adipose cells to adaptation, injury, and disease. In particular, we are interested in the following topics:

Transcriptional regulation of skeletal muscle gene expression in response to exercise and disuse.

Transcriptional regulation of adipocyte gene expression.

The role of various signaling pathways involved in muscle hypertrophy and atrophy.

The effects of psychological stress and obesity on muscle and skeletal muscle and adipose gene expression.

Molecular mechanisms underlying exercise-induced muscle damage and repair.

We employ a broad range of integrative techniques and approaches to study these questions. The techniques include genetic cloning of regulatory regions, untranslated regions, and coding regions for cellular and in vivo over-expression studies; mutagenesis and deletional analysis of genes and their regulatory regions; regular and real-time RT-PCR; immunohistochemistry, ELISA and Western blotting; cell culture; transfection of cultured fat and muscle cell lines; luciferase and green fluorescent protein (GFP) reporter analysis; null and transgenic mouse analysis; histology and histochemistry; and voluntary cage wheel and treadmill running exercise training. Our work utilizes a highly integrative approach spanning bioinformatics and sequence analysis, cell culture, animal models, and human clinical studies.

Personnel

Director: David L. Allen, Ph.D.

Undergraduate Students: Brianna Fowler, Stephen Nikaido, Jillian Warner, Sherry Wang, Cameron Niswander.

Graduate Students: Ryan Mehan, Molly Madden.

Collaborators: Dr. Virginia Ferguson and Dr. Louis Stodiek, BioServe Space Technologies, Aerospace Engineering, CU-Boulder.

Current Research Projects

Regulation of myostatin expression and function during muscle atrophy. Myostatin is a member of the TGF-beta family of growth factors that is expressed exclusively in skeletal muscle and which functions to inhibit muscle growth and development. The factors regulating myostatin expression in skeletal muscle are currently not well defined; our goal is to identify the cis- and trans-regulatory elements and the signaling pathways involved in myostatin gene transcription, and furthermore to explore whether pathophysiological states such as stress, food deprivation, obesity or unloading affect myostatin expression in mice. We are using transfection of human and mouse myostatin promoter constructs into cultured muscle cells, along with co-transfection with signaling and transcription factor expression vectors and mutagenesis, to address the role of transcriptional mechanisms in regulating myostatin expression. In addition, we have recently identified a well-conserved microRNA pathway which appears to regulate myostatin mRNA stability post-transcriptionally.

Regulation of interleukin-6 (IL-6) expression in skeletal muscle in response to exercise. IL-6 is a cytokine whose expression by and secretion from skeletal muscle is hugely increased following a single bout of endurance exercise. IL-6 is thought to function as an "energy sensor", linking an increase in muscle activity with the mobilization of glucose and lipid stores from liver and fat during prolonged exercise. It is currently not known what the signal is for the increase in skeletal muscle IL-6 expression in response to exercise. The goal of this project is to identify the transcriptional and/or post-transcriptional regulatory elements, and their associated signaling pathways, involved in activation of IL-6 expression in response to exercise. For this study, the human and mouse IL-6 promoter and untranslated regions have been cloned into reporter constructs in order to determine the sequence(s) necessary for this systems level response. Both cell culture and animal studies are being used to identify the mechanisms regulating IL-6 expression under basal and exercise-stimulated conditions. In addition, human studies will be run in which subjects with specific IL-6 promoter and UTR genotypes are exercised to see whether naturally occurring polymorphisms in the human IL-6 regulatory regions influence the magnitude of the exercise IL-6 response.

Role of matrix metalloproteinases in exercise-induced muscle damage and repair. A single bout of eccentric exercise often causes muscle damage and soreness for days afterward. Currently the molecular etiology of this process is not well defined. The goal of this project is to determine the role, if any, of connective tissue-degrading enzymes known as matrix metalloproteinases or MMPs in the exercise-induced damage/repair process. MMPs are known to be secreted by many cell types involved in muscle damage and repair, including fibroblasts, immune cells, and the muscle fibers themselves. We will explore the causal role of MMPs in exercise-induced damage and repair by inhibiting MMP activity pharmacologically and MMP expression genetically in eccentrically-exercised mice. In addition, the factors regulating MMP transcription will be identified using MMP promoter constructs transfected into C2C12 myotubes in vitro and injected into mouse skeletal muscles in vivo. Finally, human clinical studies will determine whether serum MMP levels mimic the serum levels of other indices of muscle damage in eccentrically-exercised human subjects.

Opportunities for Undergraduates

Dr. Allen is no longer accepting undergraduate researchers for the 2011-2012 academic year.

Funding

2008-2011, NIH/NIAMS R03, "Regulation of Exercise-Induced IL-6 Expression in Skeletal Muscle".

2009-20011, University of Colorado Innovative Seed Grant, "Cellular and Molecular mechanisms underlying skeletal muscle changes in response to acute and chronic psychological stress".