BADERC- Herbert Y Lin

Herbert Y Lin, MD, Ph.D.

TGF-b Signaling and Diabetic Nephropathy

Use of Soluble TGF-b Receptors in Treatment of Diabetic Nephropathy. Diabetes mellitus is one of the most common causes of end-stage renal disease, accounting for up to one-third of all patients requiring hemodialysis (USRDS 1999). The pathology of diabetic nephropathy is characterized by thickening of the glomerular basement membrane, mesangial cell proliferation, accumulation of mesangial extracellular matrix with blockage of the glomerular capillary lumen with eventual loss of glomerular function and the clinical onset of proteinuria, hypertension, and kidney failure. The mechanisms leading to the development of renal dysfunction and the characteristic structural abnormalities seen in diabetic nephropathy are not well understood. More recently, an expanding body of evidence that elevated transforming growth factor b (TGF-b) expression may play an important role in the pathogenesis of diabetic renal disease has accumulated. However, the exact roles and relative contributions to pathology of each isoform (TGF-b1, -b2, or –b3) has not been addressed.

Most mammalian cells express three abundant high affinity receptors which can bind and be cross-linked to TGF-b: the type I (~53kDa), type II (~65 kDa), and the type III (~100-280 kDa) receptors. While TGF-b1 binds with high affinity (50-500 pM) to the type I, II and III receptors at the cell surface, TGF-b2 binds with high affinity only to the type III receptor while binding poorly to the majority of the type I and type II receptors. The type II receptor and type I receptors are transmembrane proteins with cytosolic domains containing serine/threonine kinases homologous to that found in the activin and several other receptors. The type III receptor is a membrane bound proteoglycan with a short cytoplasmic tail that has no apparent signaling motif.

We have engineered soluble extracellular domain TGF-b type II and type III receptors fused to Fc. We show that the sTbRIII∆.Fc (lacking GAG attachment sites) has high affinity for TGF-b2, as well as for TGF-b1 and -b3. For this reason, we chose the sTbRIII∆.Fc as our receptor to neutralize TGF-b2 signals. We have also shown that the sTbRII-Fc protein can bind TGF-b1 and TGF-b3 but not TGF-b2. Thus, we will compare the effect of treatment of db/db diabetic mice with either sTbRIII∆.Fc or sTbRII-Fc (“sparing” of TGF-bb) to assess the specific role of TGF-b2 in the development of diabetic nephropathy.

References:

1. Roelen BA, Cohen OS, Raychowdhury MK, Chadee DN, Zhang Y, Kyriakis JM, Alessandrini AA, Lin HY. (2003). Phosphorylation of threonine276 in Smad4 is involved in transforming growth factor-b-induced nuclear accumulation. Am J Physiol Cell Physiol. June 11, 2003. [Epub ahead of print]

2. Wells, R. G., Yankelev, H., Lin, H.Y., and H.F. Lodish. (1996). Biosynthesis of the type I and type II TGF-b receptors: implications for complex formation. J. Biol. Chem.

272:11444-11451.

3. Moustakas, A., Takumi, T., Lin, H.Y., and H.F. Lodish. (1995). GH3 pituitary tumor cells contain heteromeric type I and type II receptor complexes for transforming growth factor b and activin A. J. Biol. Chem. 270:765-769.

4. Lin, H.Y., Moustakas, A., Knaus, P., Wells, R. G., Henis, Y. I., and H.F. Lodish. (1995). The soluble exoplasmic domain of the type II transforming growth factor (TGF)- b receptor. J. Biol. Chem. 270:2745-2747.

5. Takumi, T., Moustakas, A., Lin, H.Y., and H.F. Lodish. (1995). Molecular characterization of a type I serine-threonine kinase receptor for TGF-b and activin in the rat pituitary tumor cell line GH3. Exp. Cell Res. 216:208-214.

6. Lin, H.Y., and A. Moustakas. (1994). TGF-b receptors: structure and function. Cell. Mol. Biol. 40:337-349.

7. Lin, H.Y. and X.-F. Wang (1992). Expression cloning of TGF-b receptors. Molec. Reprod. and Develop. 32:105-110.

8. Lin, H.Y., and H. F. Lodish (1993). Receptors for the TGF-b superfamily: multiple polypeptides and serine/threonine kinases. Trends in Cell. Biol. 3:14-19.

9. Lin, H.Y., Wang, X-F., Ng-Eaton, E., Weinberg, R. A., and H. F. Lodish (1992). Expression cloning and characterization of the TGF-b type II receptor, a functional transmembrane serine/threonine kinase. Cell 68:775-785.

10. Wang, X-F., Lin, H.Y., Ng-Eaton, E., Downward, J., Lodish, H. F., and R. A. Weinberg. (1991).Expression cloning and characterization of the type III TGF-b receptor. Cell 67:797-805.

11. del Re E, Babitt JL, Pirani A, Schneyer AL, Lin HY. In the absence of type III receptor, the transforming growth factor (TGF)-beta type II-B receptor requires the type i receptor to bind TGF-beta2. J Biol Chem. 2004;279:22765-72.






			Herbert Y Lin, MD, Ph.D. TGF-b Signaling and Diabetic Nephropathy Use of Soluble TGF-b Receptors in Treatment of Diabetic Nephropathy. Diabetes mellitus is one of the most common causes of end-stage renal disease, accounting for up to one-third of all patients requiring hemodialysis (USRDS 1999). The pathology of diabetic nephropathy is characterized by thickening of the glomerular basement membrane, mesangial cell proliferation, accumulation of mesangial extracellular matrix with blockage of the glomerular capillary lumen with eventual loss of glomerular function and the clinical onset of proteinuria, hypertension, and kidney failure. The mechanisms leading to the development of renal dysfunction and the characteristic structural abnormalities seen in diabetic nephropathy are not well understood. More recently, an expanding body of evidence that elevated transforming growth factor b (TGF-b) expression may play an important role in the pathogenesis of diabetic renal disease has accumulated. However, the exact roles and relative contributions to pathology of each isoform (TGF-b1, -b2, or –b3) has not been addressed. Most mammalian cells express three abundant high affinity receptors which can bind and be cross-linked to TGF-b: the type I (~53kDa), type II (~65 kDa), and the type III (~100-280 kDa) receptors. While TGF-b1 binds with high affinity (50-500 pM) to the type I, II and III receptors at the cell surface, TGF-b2 binds with high affinity only to the type III receptor while binding poorly to the majority of the type I and type II receptors. The type II receptor and type I receptors are transmembrane proteins with cytosolic domains containing serine/threonine kinases homologous to that found in the activin and several other receptors. The type III receptor is a membrane bound proteoglycan with a short cytoplasmic tail that has no apparent signaling motif. We have engineered soluble extracellular domain TGF-b type II and type III receptors fused to Fc. We show that the sTbRIII∆.Fc (lacking GAG attachment sites) has high affinity for TGF-b2, as well as for TGF-b1 and -b3. For this reason, we chose the sTbRIII∆.Fc as our receptor to neutralize TGF-b2 signals. We have also shown that the sTbRII-Fc protein can bind TGF-b1 and TGF-b3 but not TGF-b2. Thus, we will compare the effect of treatment of db/db diabetic mice with either sTbRIII∆.Fc or sTbRII-Fc (“sparing” of TGF-bb) to assess the specific role of TGF-b2 in the development of diabetic nephropathy. References: 1. Roelen BA, Cohen OS, Raychowdhury MK, Chadee DN, Zhang Y, Kyriakis JM, Alessandrini AA, Lin HY. (2003). Phosphorylation of threonine276 in Smad4 is involved in transforming growth factor-b-induced nuclear accumulation. Am J Physiol Cell Physiol. June 11, 2003. [Epub ahead of print] 2. Wells, R. G., Yankelev, H., Lin, H.Y., and H.F. Lodish. (1996). Biosynthesis of the type I and type II TGF-b receptors: implications for complex formation. J. Biol. Chem. 272:11444-11451. 3. Moustakas, A., Takumi, T., Lin, H.Y., and H.F. Lodish. (1995). GH3 pituitary tumor cells contain heteromeric type I and type II receptor complexes for transforming growth factor b and activin A. J. Biol. Chem. 270:765-769. 4. Lin, H.Y., Moustakas, A., Knaus, P., Wells, R. G., Henis, Y. I., and H.F. Lodish. (1995). The soluble exoplasmic domain of the type II transforming growth factor (TGF)- b receptor. J. Biol. Chem. 270:2745-2747. 5. Takumi, T., Moustakas, A., Lin, H.Y., and H.F. Lodish. (1995). Molecular characterization of a type I serine-threonine kinase receptor for TGF-b and activin in the rat pituitary tumor cell line GH3. Exp. Cell Res. 216:208-214. 6. Lin, H.Y., and A. Moustakas. (1994). TGF-b receptors: structure and function. Cell. Mol. Biol. 40:337-349. 7. Lin, H.Y. and X.-F. Wang (1992). Expression cloning of TGF-b receptors. Molec. Reprod. and Develop. 32:105-110. 8. Lin, H.Y., and H. F. Lodish (1993). Receptors for the TGF-b superfamily: multiple polypeptides and serine/threonine kinases. Trends in Cell. Biol. 3:14-19. 9. Lin, H.Y., Wang, X-F., Ng-Eaton, E., Weinberg, R. A., and H. F. Lodish (1992). Expression cloning and characterization of the TGF-b type II receptor, a functional transmembrane serine/threonine kinase. Cell 68:775-785. 10. Wang, X-F., Lin, H.Y., Ng-Eaton, E., Downward, J., Lodish, H. F., and R. A. Weinberg. (1991).Expression cloning and characterization of the type III TGF-b receptor. Cell 67:797-805. 11. del Re E, Babitt JL, Pirani A, Schneyer AL, Lin HY. In the absence of type III receptor, the transforming growth factor (TGF)-beta type II-B receptor requires the type i receptor to bind TGF-beta2. J Biol Chem. 2004;279:22765-72.