Tien C. Ko, MD: TGF-beta Signaling Pathways: From the Receptor to the Nucleus

This morning I'd like to focus my talk on an area of our interest in the lab, that's in transforming growth factor beta.

[ Slide 01 ]   Transforming Growth Factor beta (TGF-beta)

Transforming growth factor beta is a 25 kD homodimeric protein with three mammalian isoforms, type 1, 2 and 3. It's a member of the TGF-beta superfamily of peptides, which includes activin, bone morphogenic, protein, and others. These peptides are important regulators of diverse biologic processes, including proliferation, differentiation and apoptosis, and as a result they have generated quite a bit of interest for investigators.

[ Slide 02 ]   TGF-beta and the Gut

Since I'm a general surgeon, I'm particularly interested in the gut and as far as TGF-beta in the gut, it is secreted by the enterocytes in the villus compartment of the gut. It inhibits proliferation and stimulates migration and differentiation of gut epithelial cells. It promotes restitution of intestinal epithelium following injury. So TGF-beta appears to have both a physiologic role and perhaps a role during pathophysiology in the gut.

[ Slide 03 ]   TGF-beta and Colorectal Cancer

More interestingly, it also has a role in colorectal cancer. There appears to be a loss of TGF-beta responsiveness during malignant transformation of colorectal cancer. TGF-beta receptor is deleted in a subset of colon cancer therefore blocking the signal transduction pathways. Recently it's been shown that deletion of the TGF-beta signaling protein leads to an animal model of colorectal cancer in a knockout mouse. So these data certainly highlight the importance of the role of TGF-beta in the pathogenesis of colorectal cancer.

[ Slide 04 ]   The TGF-beta Signaling Pathway

Over the last 10 years, the TGF-beta pathway has been studied by a number of labs, and it's pathway has been delineated as shown here. The TGF-beta peptide binds to its receptors... In this case there are two receptors, there's a Type 2 receptor, R II, and a Type 1 receptor. They form a complex and the Type 2 receptor activates the Type 1 receptor by phosphorylating it's amino acids. The activated Type 1 receptor then recruits a signaling molecule, which is a transcription factor, called the Smad family of proteins. The Smad proteins are phosphorylated, forms a multimeric complex that then enter the nucleus leading to its effect. And in this case, what we are particularly interested in is in cell cycle arrest.

[ Slide 05 ]   Flowcytometry

Using a rat intestinal epithelial cell line, what we've shown is that when you treat these cells with TGF-beta it exerts a G1 block. When these cells are allowed to enter the S phase, in the presence of TGF-beta, there's a significant suppression of cells in the S phase, while in the absence, these cells go on to cell cycle progression.

[ Slide 06 ]   G1 Cell Cycle Control

The question is, what is the mechanism involved? Well, as you heard a little bit from Dr. Hunt, about the cyclin, Cdk, as well as the Rb pathway, it's delineated here in terms of the G1 cell cycle control. The key component of the cell cycle control is the cyclin/Cdk complex. The cyclin proteins, cyclin D1, 2 and 3 form a complex with its kinase partner, Cdk 4 or 6. This activated kinase is able to phosphorylate key substrates, one of them is the Rb protein. In its under-phosphorylated form, the Rb protein is able to bind E2F leading to repression of E2F activity. Upon phosphorylation, the Rb protein becomes inactive and E2F then goes on to stimulate gene expression and the cell is allowed to progress from G1 into S.

[ Slide 07 ]   TGF-beta in the Signaling Pathway - 1

So we began looking at the effect of TGF-beta in the signaling pathway of the cell cycle by looking at RNA expression of cyclin D1, 2, 3 and the catalytic partner Cdk4 by Northern blot. As you can see, treatment with TGF-beta lead to decrease in cyclin D1 expression, in the two intestinal epithelial cell line, IEC-6 and RIE-1, while there is really no effect on cyclin D2, D3 or Cdk4.

[ Slide 08 ]   TGF-beta in the Signaling Pathway - 2

Next, we determined whether this decrease in message leads to a decrease in protein expression. This is a Western blot, showing a decrease in cyclin D1 expression at the protein level, while D2, D3 and Cdk4 had no effect.

[ Slide 09 ]   Metabolic Labeling Studies

We wanted to know the mechanism involved in the down-regulation of cyclin D1 protein, so we did metabolic labeling studies, to determine whether there's inhibition in protein synthesis, an increase in protein degradation. In panel B is a metabolically labeled experiment in which we see there's a decrease in cyclin D1 synthesis following TGF-beta treatment while Cdk4 synthesis remained unchanged. Using a metabolic pulse-chase experiment, we also determine the half-life of cyclin D1 protein following TGF-beta treatment. As you can see, there's no change compared to the control. So the mechanism involved in down-regulation of d1 protein expression is due to a decrease in protein synthesis.

[ Slide 10 ]   Co-IP Experiment

As I mentioned before, cyclin D1 forms complex with its partner Cdk4. So next we asked whether the decrease in D1 expression leads to a decrease in cyclin D/Cdk4 complex formation. To do that, we did a co-IP experiment. We collected cell lysates and immunoprecipitated Cdk4 complexes with Cdk4 antibody and determined how much cyclin D1 is present in that complex. As you can see, in the presence of TGF-beta there's a decrease in cyclin D/Cdk4 complex. We did a reciprocal experiment and we got the same result. Not only do we want to know whether the complex is decreased, the most important thing is whether the activity of Cdk4 is decreased. To do that we use a recombinant protein Rb and to ask whether Cdk phosphorylation of this protein is decreased following TGF-beta treatment. As you can see there's a dramatic decrease in Cdk4 kinase activity following TGF-beta treatment.

[ Slide 11 ]   Cyclin d1 in Cell Cycle Progression

If cyclin D1 is the target of TGF-beta and if it's related to cell cycle arrest, then another method of decreasing cyclin D1 should lead to similar results, that is cell cycle arrest. So we used a strategy of a antisense oligonucleotide that's targeted against the 5' region of cyclin D1. And treatment of cells in lane 5 with D1 antisense oligonucleotide dramatically decrease the expression of cyclin D1 back to control levels and similarly it blocked progression of cell cycle down to control level indicating that cyclin D1 is important in our cells for cell cycle progression.

[ Slide 12 ]   Can Over Expression of Cyclin D1 Block TGF-beta

We then asked whether over expression of cyclin D1 can block the effects of TGF-beta. So we generated a cell line that has an inducible expression of human cyclin D1. As you can see that inducible system is tetracycline. In the presence of tetracycline, the human D1 gene is turned off by the inducible promoter. In the absence of tetracycline, there's expression of human D1, both at an mRNA and protein level.

[ Slide 13 ]   Tet-d1

And when we took these cells, which we call tet-D1, and test for their resistance to TGF-beta, you can see that over expression of human cyclin D1 lead to increase in cells in the S phase, even in the presence of TGF-beta suggesting that D1 is indeed the target and the cause of cell cycle arrest in these cells.

[ Slide 14 ]   Proliferation Assay - 1

Recently we started focusing on the signaling molecules of TGF-beta, that's alluded to by Dr. Berger as the Smad family of proteins and what we first wanted to focus on is to determine whether Smad proteins are rate limiting in the signaling of TGF-beta. So we generated a cell line that overexpressed Smad 3 protein using a retrovirus vector. Over expression of Smad 3 enhanced TGF-beta suppression of growth compared to control cell line that's just infected with just a vector retrovirus. So we can see that indeed by giving the cell more Smad 3, they now are more susceptible to the negative effect of TGF-beta.

[ Slide 15 ]   Proliferation Assay - 2

We then did a double infection of these cell lines by co-infecting the Smad 3 with a R2 dominated negative mutant. That's a Type 2 receptor that's truncated of it's cytoplasmic domain so it no longer is able to transduce a signal. When this cell line is treated with TGF-beta, we no longer see the suppressor effect of TGF-beta, again indicating that you need both the receptor and the Smad protein for signaling.

[ Slide 16 ]   Summary

So this is a summary of what I present. Our working hypothesis is that TGF-beta, through its receptor, Type 2 and Type 1 receptor, is able to recruit Smad protein leading to, in our case, cell cycle arrest by targeting the cyclin D/Cdk4 complex.

[ Slide 17 ]   Current Research/Goals

And our current research and goals are to delineate the roles of the Smad molecules during TGF-beta signaling in the gut and we want to identify other signaling pathways that may interact with TGF-beta to regulate gut physiology. Our ultimate goal is to increase our understanding of the signaling pathway that governs both normal and malignant gut epithelial cell proliferation. When we can identify these pathways then we can perhaps generate models for colon diseases as well as colon cancer.

[ Slide 18 ]   Acknowledgement

Lastly I want to thank my research staff as well as collaborators under the direction of Dr. Townsend and our research people in my lab as well as Aubrey Thompson in the basic science department, Dr. Beauchamp at Vanderbilt and recently collaboration with UCSF with Dr. Derynck's lab where I did a year of sabbatical. Thank you.