Robbins Lab

Molecular Studies of Human Valve Disease

Figures

Structure of SHP-2 and One of its Roles in Cell Signaling

Structure of SHP-2 and One of its Roles in Cell Signaling.

A. Shown is the gene structure of PTPN11 and relevant domains of the protein, which are closely conserved between mouse and human. The corresponding exons that encode the domain are indicated, as are a few of the characterized mutations (in red). *A mutation targeted for study in this application, Gln79->Arg, is located in the NH3 SH2 domain and is found frequently in NS patients (15).

B. FGFR signaling as an example. A role for SHP-2 in myocyte differentiation has also been outlined, possibly via signaling through the fibroblast growth factor receptor (FGFR) pathway. Stimulation of the FGFR in myoblasts (and perhaps in cardiomyocytes) prevents or slows myocyte differentiation and over-expression of either wild type or constitutively active forms of SHP-2 in the skeletal myoblast line C2C12 also prevents differentiation. Activation of FGFR leads to tyrosyl phosphorylation of FRS2, a lipid-anchored, adaptor molecule that recruits the SH2 domain-containing protein Grb2, and SHP-2. Grb2, itself, can also bind to SHP-2, when SHP-2 residues Y542,580 becomes tyrosyl-phosphorylated in response to growth factor receptor activation. This tyrosyl phosphorylation creates a Grb2 binding site and promotes assembly of the protein complex, linking receptor activation to the ERK pathway.

The myosin light chain promoter will be used to drive SHP-2 expression during embryogenesis

The myosin light chain promoter will be used to drive SHP-2 expression during embryogenesis.

Developmental expression of MLC2v and MLC2v-CAT. (A, B) Endogenous MLC2v expression detected by whole mount in situs at E8.5 (A) and E9.5 (B). MLC2v is restricted to the anterior ventricular heart tube at E8.5 and the ventricles and A-V junction at E9.5. (->, the ventricles; arrowheads, the atria.) (C, D) Immunolocalization of CAT protein expression in MLC2v-CAT transgenic embryo at E9.5 (C) and adult (D). Embryonic expression of MLC2v-CAT, like the endogenous gene, predominates in the ventricular and A-V junction segments (E9.5). ot; outflow tract.

Mutations in PTPN11 cause Noonan syndrome. The lab is exploring the mechanistic underpinnings of PTPN11's role in valve formation. The objective is to mechanistically dissect the cardiac - autonomous pathologies of Noonan syndrome. Our immediate goals are to carry out comprehensive studies using inducible, cardiac-specific expression of both the normal and mutated forms of the tyrosine phosphatase, SHP-2 in the different cardiac cell populations. These studies will be complemented by an inducible, cardiac-specific gene ablation of PTPN11 in order to discern protein function at different developmental times. SPECIFIC AIM 1 will explore the cardiomyocyte autonomous effects of SHP-2 expression in order to dissect the primary and secondary effects on hypertrophy and valve dysfunction. Wild type (WT) and mutated protein will be expressed only in the cardiomyocyte population via transgenesis. The hypothesis is that expression of the Noonan mutation SHP-2Gln79Arg, will result in cardiomyocyte hypertrophy. SPECIFIC AIM 2 will carry out the complementary studies in the relevant non-cardiomyocyte populations to define the role that the SHP-2 mutation plays during cardiac cushion formation and development of the outflow tract. Both the WT and mutated protein will be expressed during development in the endothelial population only. SPECIFIC AIM 3 will explore loss of function of the normal protein by carrying out an inducible, cardiomyocyte-specific knockout using the MerCreMer system developed in our Division. We hypothesize that the effects of SHP-2 loss of function will differ radically depending upon the developmental time and in this manner the role of SHP-2 in controlling normal cellular processes in the heart can be explored. SPECIFIC AIM 4 will explore the signaling pathways downstream of SHP-2 in cardiomyocytes. We hypothesize that SHP-2 signals through activation of the MAP kinase (MAPK) pathway in cardiomyocytes and that ablation of SHP-2 will blunt MAPK signaling while over-expression of WT-SHP-2 or SHP-2Gln79Arg will result in increased MAPK activity or inappropriate MAPK activity through the ERK branch in response to various stimuli.

Related Publications

Nakamura T, Colbert M, Krenz M, Molkentin JD, Hahn HS, Dorn II GW, Robbins J. (2007) Mediating ERK1/2 signaling rescues congenital heart defects in a mouse model of Noonan syndrome. J Clin Invest 117: 2123-2132.

Hsieh PCH, Davis ME, MacGillivray C, Gannon J, Molkentin JD, Robbins J, Lee RT. (2007) Evidence that stem cells refresh adult mammalian cardiomyocytes following injury: A genetic fate-mapping study. Nature Med 13:970-974.

Nakamura T, Colbert MC, Robbins J. (2006) Neural crest cells retain multipotential characteristics in the developing valves and label the cardiac conduction system. Circ Res 98:1547-1554.

Krenz M, Yutzey KE, Robbins J. (2005) Noonan syndrome mutation Q79R in Shp2 increases proliferation of valve primordia mesenchymal cells via extracellular signal-regulated kinase 1/2 signaling. Circ Res 97:813-20.

Yutzey KE, Colbert M, Robbins J. (2005) Ras-related signaling pathways in valve development: ebb and flow. Physiology (Bethesda) 20:390-7.

Sanbe, A., Gulick, J., Hanks, M. C., Liang, Q., Osinska, H., Robbins, J. (2003) Reengineering inducible cardiac-specific transgenesis with an attenuated myosin heavy chain promoter. Circ. Res. 92:609-616.

Schwartzbauer. G., Robbins, J. (2001) The tumour suppressor gene pten can regulate cardiac hypertrophy and survival. J. Biol. Chem. 276, 35786-35793

Nelson, D., P., Setser, E., Hall, D., G., Schwartz. S., M., Hewett, T. E., Klevitsky, R., Osinska, H., Bellgrau, D., Duke, R. C., and Robbins, J. (2000) Proinflammatory consequences of transgenic Fas ligand expression in the heart. J Clin Invest. 105; 1199-1203

Contact Dr. Robbins

Jeffrey Robbins
Molecular Cardiovascular Biology
ML 7020
Children's Hospital
3333 Burnet Ave
Cincinnati, OH 45229-3039
jeff.robbins@cchmc.org