Project 2:  Cell damage and remodeling pathways in DMD

Yi-Wen Chen, PhD

Kanneboyina Nagaraju, PhD

A theme that has emerged in many disorders is the extraordinary plasticity and remodeling that a tissue undergoes when faced with a pathological situation.  Muscle is normally one of the more plastic tissues, and the pathways involved in hypertrophy, atrophy, and other types of muscle remodeling are complex in their own rite.  The interactions of normal plasticity pathways, myofiber necrosis and regeneration pathways, and inflammatory pathways all lead to a highly complex process with extensive possibilities for cross-talk between pathways.

We have chosen three specific types of pathways for “dissection” in Project 2 of the Center.  Two of these pathways are shared among most types of tissue damage; namely the acute response of the Toll-like receptors (TLRs), NFkappaB, and inflammatory mediators, and the more chronic response of the TGF-beta pathways.  The Center investigators are fortunate in having considerable resources that are not available elsewhere, and these are used to begin to dissect these complex pathways, and to define cross-talk between them.  First, we are fortunate in having an extensive tissue bank of flash frozen muscle.  The Hoffman lab has run a for gratis molecular diagnostic service since the original discovery of dystrophin in 1987, with the “deliverable” of a 5,000+ muscle biopsy tissue bank.  All biopsies have been histologically analyzed, and all clinical, biochemical testing, and histological assessments entered into a laboratory database.  This database can be queried with patient identifiers blocked, and the resulting biopsies accessed for research studies.  All subjects are covered under IRB approvals that have followed the tissue bank and database, from Children’s Boston, to Pittsburgh, and now Washington DC.  These biopsies include hundreds of well preserved, dystrophin-validated, Duchenne muscular dystrophy biopsies, ranging from fetal through late teens.  This resource has permitted a “developmental series” of mRNA profiles (fetal, infant [6 month olds from neonatal screening], and symptomatic DMD patients).  Thus, the tissue bank has been converted into a genome-wide expression profiling resource, with over 800 human muscle profiles generated by the Research Center for Genetic Medicine.

The combination of tissue resource, and expression profiling expertise, has led to the hypothesis generation underlying Project 2.  We show that the TLR/NFkappaB/inflammatory pathways are already strongly activated by 6 months of age, even though patients will not show symptoms for many years.  These pathways are part of effective tissue remodeling and repair.  However, we and others have also shown that dystrophin-deficient myofibers are painfully sensitive to inflammatory mediators; what is a reparative inflammatory mediator in normal muscle likely becomes a destructive agent in dystrophin-deficient muscle.To better dissect this complex cascade, we have recruited our long-term muscle immunologist collaborator as a co-PI on Project 2, Dr. Kanneboyina Nagaraju.  Drs. Nagaraju, Hoffman, and Chen have many active projects together, ranging from polymyositis, to sexual dimorphism in immune function, to monocyte function in dysferlin deficient patients recruited into the CNMC CRC.  His research on the TLR/NFkappaB pathways is a natural extension of his previous research, as well as a new entry into research on Duchenne muscular dystrophy. 

The second pathway that is studied is the TGF-beta pathways.  TGF-β1 is a powerful and essential immune regulator, which is capable of modulating inflammatory events.  This is most dramatically illustrated by the phenotype of TGF-β1 knock-out mice which die in utero or in perinatal period because of widespread, uncontrolled inflammation.  The effect can be reversed by the systemic administration of active soluble TGF-β1.  Besides the effects on immune cells, TGF-β1 stimulates the synthesis of extracellular matrix (ECM) proteins and inhibits matrix degradation, resulting in the promotion of fibrosis and tissue repair.  In cultured muscle cell lines and primary myogenic cells, TGF-β1 suppresses both proliferation and differentiation while the insulin-like growth factors (IGF-I and IGF-II) stimulate both of these processes.  TGF-β1 was demonstrated increasing in muscles of DMD, congenital muscular dystrophy, and inflammatory myositis patients.  In this proposal, we will investigate the effect of TGF-β1 on both fibrosis and the inhibitory effect on myogenesis in mdx mice.  Dr. Yi-Wen Chen heads the research on the TGF-beta pathways in Project 2, and this is a logical extension of her work on the progression of Duchenne dystrophy at the molecular level, and her expertise in in vivo transfections using electroporation.  She shows that the TGF-beta pathways are induced relatively late in the disease process, and presents a novel model where one of the few “infant-specific” transcripts, AML1, may play an important role in suppressing the TGF-beta pathways.  We have put both the NFkappaB and TGF-beta pathways into the same project, due to the well-documented cross-talk between inflammatory cells and these two pathways.