Transforming growth factor beta (TGFbeta ) signaling via differential activation of activin receptor-like kinases 2 and 5 during cardiac development. Role in regulating parasympathetic responsiveness

Little is known regarding factors that induce parasympathetic responsiveness during cardiac development. We demonstrated previously that in atrial cells cultured from chicks 14 days in ovo, transforming growth factor beta (TGFbeta) decreased parasympathetic inhibition of beat rate by the muscarinic agonist, carbamylcholine, by 5-fold and decreased expression of Galpha(i2). Here in atrial cells 5 days in ovo, TGFbeta increased carbamylcholine inhibition of beat rate 2.5-fold and increased expression of Galpha(i2). TGFbeta also stimulated Galpha(i2) mRNA expression and promoter activity at day 5 while inhibiting them at day 14 in ovo. Over the same time course expression of type I TGFbeta receptors, chick activin receptor-like kinase 2 and 5 increased with a 2.3-fold higher increase in activin receptor-like kinase 2. Constitutively active activin receptor-like kinase 2 inhibited Galpha(i2) promoter activity, whereas constitutively active activin receptor-like kinase 5 stimulated Galpha(i2) promoter activity independent of embryonic age. In 5-day atrial cells, TGFbeta stimulated the p3TP-lux reporter, which is downstream of activin receptor-like kinase 5 and had no effect on the activity of the pVent reporter, which is downstream of activin receptor-like kinase 2. In 14-day cells, TGFbeta stimulated both pVent and p3TP-lux. Thus TGFbeta exerts opposing effects on parasympathetic response and Galpha(i2) expression by activating different type I TGFbeta receptors at distinct stages during cardiac development.     

Cirrhotic hepatocytes exhibit decreased TGFbeta growth inhibition associated with downregulated Smad protein expression

TGFbeta controls hepatocyte growth through cell cycle arrest and apoptosis, and resistance to TGFbeta is a mechanism of malignant transformation. The aim of this study was to assess differences in TGFbeta-mediated growth inhibition in normal and cirrhotic hepatocytes. Cirrhosis was induced in mice and normal and cirrhotic hepatocytes were isolated by collagenase perfusion and treated with or without TGFbeta (5 ng/ml). DNA synthesis, Smad protein expression, and DNA binding activity were determined. TGFbeta reduced DNA synthesis to a greater degree in normal hepatocytes than in cirrhotic hepatocytes (87% vs. 68%; p<0.05). Smad protein expression was decreased in cirrhotic hepatocytes and Smad 2/3/4 complex formation was suppressed. Furthermore, cirrhotic hepatocytes had decreased DNA binding activity at 120 min following TGFbeta treatment. In conclusion, decreased Smad protein expression may impair TGFbeta-mediated growth inhibition in cirrhotic hepatocytes.     

Attenuation of haptoglobin gene expression by TGFbeta requires the MAP kinase pathway.

In addition to important roles in the regulation of cell growth and cell restitution, both pro- and anti-inflammatory effects have been ascribed to TGFbeta in intestinal epithelial cells. However, the mechanisms involved in TGFbeta-dependent anti-inflammatory activities remain to be determined. In the rat intestinal epithelial cell line IEC-6, TGFbeta attenuated the glucocorticoid-dependent increases in mRNA levels of the acute phase protein gene haptoglobin, and of C/EBP isoforms beta and delta. Supershift assays demonstrated a TGFbeta-mediated decrease in the binding of C/EBP isoforms beta and delta to the haptoA and haptoC C/EBP DNA-binding sites from the haptoglobin promoter. Mutations of both HaptoA and HaptoC sites abolished the glucocorticoid-dependent activation and the TGFbeta-mediated attenuation of the haptoglobin promoter, as assessed by transient transfection assays. TGFbeta induced p42/p44 MAP kinase activities. Treatment with the MEK 1/2 inhibitor PD 98059 abolished TGFbeta attenuation. These results suggest that C/EBP isoforms are involved both in the glucocorticoid-dependent induction and in the TGFbeta-mediated attenuation of haptoglobin expression. Furthermore, p42/p44 MAP kinases may function in a TGFbeta-dependent signaling pathway leading to attenuation of haptoglobin expression.     

Activation of the cyclin D1 gene by the E1A-associated protein p300 through AP-1 inhibits cellular apoptosis

The adenovirus E1A protein interferes with regulators of apoptosis and growth by physically interacting with cell cycle regulatory proteins including the retinoblastoma tumor suppressor protein and the coactivator proteins p300/CBP (where CBP is the CREB-binding protein). The p300/CBP proteins occupy a pivotal role in regulating mitogenic signaling and apoptosis. The mechanisms by which cell cycle control genes are directly regulated by p300 remain to be determined. The cyclin D1 gene, which is overexpressed in many different tumor types, encodes a regulatory subunit of a holoenzyme that phosphorylates and inactivates PRB. In the present study E1A12S inhibited the cyclin D1 promoter via the amino-terminal p300/CBP binding domain in human choriocarcinoma JEG-3 cells. p300 induced cyclin D1 protein abundance, and p300, but not CBP, induced the cyclin D1 promoter. cyclin D1 or p300 overexpression inhibited apoptosis in JEG-3 cells. The CH3 region of p300, which was required for induction of cyclin D1, was also required for the inhibition of apoptosis. p300 activated the cyclin D1 promoter through an activator protein-1 (AP-1) site at -954 and was identified within a DNA-bound complex with c-Jun at the AP-1 site. Apoptosis rates of embryonic fibroblasts derived from mice homozygously deleted of the cyclin D1 gene (cyclin D1(-/-)) were increased compared with wild type control on several distinct matrices. p300 inhibited apoptosis in cyclin D1(+/+) fibroblasts but increased apoptosis in cyclin D1(-/-) cells. The anti-apoptotic function of cyclin D1, demonstrated by sub-G(1) analysis and annexin V staining, may contribute to its cellular transforming and cooperative oncogenic properties.     

TGFbeta signalling in control of T-cell-mediated self-reactivity

In the immune system, transforming growth factor-beta (TGFbeta) affects multiple cell lineages by either promoting or opposing their differentiation, survival and proliferation. Understanding the cellular mechanisms of TGFbeta-mediated regulation is complicated due to a broad distribution of TGFbeta receptors on the surface of different immune-cell types. Recent studies using in vivo genetic approaches revealed a critical role for TGFbeta signalling in T cells in restraining fatal autoimmune lesions. Here, we review recent advances in our understanding of a role for TGFbeta signalling in the regulation of T-cell differentiation in the thymus and in the periphery, with a particular emphasis on TGFbeta-mediated control of self-reactive T cells.     

Requirement of km23 for TGFbeta-mediated growth inhibition and induction of fibronectin expression

We previously identified km23 as a novel TGFbeta receptor-interacting protein. Here we show that km23 is ubiquitously expressed in human tissues and that cell-type specific differences in endogenous km23 protein expression exist. In addition, we demonstrate that the phosphorylation of km23 is TGFbeta-dependent, in that EGF was unable to phosphorylate km23. Further, the kinase activity of both TGFbeta receptors appears to play a role in the TGFbeta-mediated phosphorylation of km23, although TGFbeta RII kinase activity is absolutely required for km23 phosphorylation. Blockade of km23 using small interfering RNAs significantly decreased key TGFbeta responses, including induction of fibronectin expression and inhibition of cell growth. Thus, our results demonstrate that km23 is required for TGFbeta induction of fibronectin expression and is necessary, but not sufficient, for TGFbeta-mediated growth inhibition.