PodcastCell Biology

Science Signaling Podcast: 02 December 2008

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Science Signaling  02 Dec 2008:
Vol. 1, Issue 48, pp. pc13
DOI: 10.1126/scisignal.148pc13

Abstract

This is a conversation with Sam Lee and Pat Ongusaha, authors of a Research Article published in the 25 November issue of Science Signaling. Drs. Lee and Ongusaha discuss the connection between Rho-associated kinase 1 (ROCK1) and Jun N-terminal kinase (JNK) signaling in the apoptotic response to ultraviolet B irradiation.

(Length: 12 min; file size: 5.72 MB; file format: mp3; location: http://podcasts.aaas.org/science_signaling/ScienceSignaling_081202.mp3)

Technical Details

Length: 12 min

File size: 5.72 MB

File Format: mp3

RSS Feed: http://stke.sciencemag.org/rss/podcast.xml

Download Podcast: http://podcasts.aaas.org/science_signaling/ScienceSignaling_081202.mp3

Educational Details

Learning Resource Type: Audio

Context: High school upper division 11-12, undergraduate lower division 13-14, undergraduate upper division 15-16, graduate, professional, general public and informal education

Intended Users: Teacher, learner

Intended Educational Use: Learn, teach

Discipline: cell biology,

Keywords: Science Signaling, signal transduction, ultraviolet light, UVB, radiation, DNA damage, apoptosis, JNK, ROCK1, Rho, kinase, JIP-3, MAPK

Transcript

Host – Annalisa VanHookWelcome to the Science Signaling Podcast for December 2nd, 2008. I’m Annalisa VanHook. In this episode we’ll be discussing a paper published in the November 25th issue of Science Signaling about how skin cells respond to ultraviolet, or UV, radiation (1). UV radiation can cause DNA damage which, in turn, can lead to cancer. And one of the ways the body tries to prevent this is by inducing the cells that have been exposed to high levels of UV to kill themselves. The work we’ll be talking about was performed by Dr. Pat Ongusaha in the laboratory of Dr. Sam Lee at Harvard Medical School. I spoke with both Dr. Ongusaha and Dr. Lee about their work.

Dr. Lee, thank you for talking with me.

Interviewee – Sam LeeThank you for having me.

Interviewer – Annalisa VanHookYour paper is about UVB damage to cells. Before we talk about the results of your work, could you tell us what sort of damage ultraviolet light causes and how this damage could lead to cancer, for example?

Interviewee – Sam LeeSure. The ultraviolet damage can cause two different damage: first with oxidative stress damage, which cause DNA modification and some DNA base loss and strand break that end up leading to gene mutation and then that cause tumor. And the other damage also caused by UV, ultraviolet, is DNA damage. It’s very common that for these carcinogen cause, so DNA damage can also cause single-stranded break in the DNA and then formation of pyrimidine dimer and thymidine dimers so that cause gene mutation and then cause tumor.

Interviewer – Annalisa VanHookSo, how does the body identify cells that have been damaged by UV, and does it try to repair them?

Interviewee – Sam LeeRight, exactly, so normally there is not excessive damage that there’s a repair mechanism can be activated that first the protein gets activated—people believe that p53 tumor suppressor and ATM pathway—that activated then some of the repair machinery can be activated then repair DNA damage. However, depends on some people, there are some people more sensitive to this DNA damage and then they can make excessive DNA damage to the cells. So in that case, the cell has to suicide and they call apoptosis. So, that’s one of the defense mechanisms that we have.

Interviewer – Annalisa VanHookHow do cells initiate apoptosis?

Interviewee – Sam LeeSo, the cell can sense this DNA damage we just described that is excessive DNA damage there that then the apoptosis mechanism can be activated. So far the apoptosis, there are two different apoptosis mechanisms there: intrinsic, extrinsic. Intrinsic is through the mitochondria. So, mitochondrial protein can be activated so they can trigger this cell death pathway. The other pathway is called extrinsic pathway through the cell-death receptor pathway, it is outside of cell membrane there is some receptor can be sensing this pathway. The main example of this type of receptor is the TNF receptor pathway. So, mainly, however, the UV-mediated DNA damage apoptosis is through this intrinsic mitochondrial-mediated cell death pathway.

So, that JNK’s JNK signaling, one of these major mitochondrial cell death pathway, which can be activated through this pathway.

Interviewer – Annalisa VanHookUltraviolet radiation comes from sunlight. How much UV radiation do we get, for example, from sunlight? Or are there any sources of UV radiation that we’re regularly exposed to?

Interviewee – Sam LeeSo, normally from sunlight, solar light, there’s three different UV lights we can have here. One is UVA, UVB, UVC. So, UVA in this represented most ultraviolet we are getting from the sun. UVC, which is most of light, can be absorbed by ozone layer. So, UVA and UVB is the major source of light we getting from the sun to the Earth. So 90% of this UV is coming from UVA and then about 10% UVB reach to the surface. So, basically UVA and UVB is the main component of these UV light from the sun can affect us as a potential carcinogen.

Interviewer – Annalisa VanHookSo, do all three types of ultraviolet radiation then have the potential to cause DNA damage or other…

Interviewee – Sam LeeExactly, all of them can cause DNA damage leading to gene mutations. However, UVC is short wavelength, about 180 to 280, very short, that can be absorbed by ozone layer. So, basically UVA and UVB are the major carcinogenic components of sunlight and related to tumor and skin cancer.

Interviewer – Annalisa VanHookHow did you identify the components that connect Jun kinase signaling to UV-induced apoptosis?

Interviewee – Sam LeeWe’ve been working on Rho kinase signaling for last few years, and we’ve been interested in how that Rho kinase activated and what’s the downstream target response, the DNA damage response, and then that end up causing apoptosis. So, what we did is—we didn’t know at that time that Jun kinase is really activated through that Rho kinase—so what we did is use TAP technology, is kind of proteomics approach. You know asking what really protein the cells binds to the Rho kinase. So, using this protein technology, we identified that one of the proteins involved in Jun kinase is called JIP-3. Basically that stands for Jun kinase interacting protein number three. So, we got a clue that Rho kinase have some connection to the Jun kinase signaling. Jun kinase has been known for number of years that it has an important role in the cell death pathway and also DNA damage-mediated apoptosis.

Interviewer – Annalisa VanHookSo, you used this Rho kinase which you had previously identified as being involved in the response to UV damage to find out what proteins function downstream of it and you identified JIP-3 which functions in Jun kinase signaling. How does JIP-3 function in Jun kinase signaling and does it function to connect the UV response to apoptosis?

Interviewee – Sam LeeRight, that’s a good question. It’s been known that JIP-3 is essential for the Jun kinase activation, that previously some investigators identified that JIP-3 as scaffolding protein binds to Jun kinase. That is essential for Jun kinase downstream Jun kinase activation. So based on that, what we found that is Rho kinase directly binds to JIP-3 and phosphorylation of JIP-3 that is important for Jun kinase activation—actually required for Jun kinase activation, that’s what we are finding to begin with. People found a few years back that JIP-3 also binds to Jun kinase and that’s necessary for Jun kinase activation; however, upstream of the JIP-3 is a black box. So people is wondering how JIP-3 binds to that and then how that Jun kinase activation in response to DNA damage is unclear.

Interviewer – Annalisa VanHookHello, Dr. Ongusaha

Interviewee – Pat OngusahaHi.

Interviewer – Annalisa VanHookDr. Ongusaha then told me about how she did the research that’s described in the paper. So, how did you identify JIP-3 as playing a role in inducing the apoptotic response to UV damage?

Interviewee – Pat OngusahaWe identified JIP-3 through tandem affinity protein purification method and we found JIP-3, JNK scaffolding protein, is present in our TAP purification. And then we also identified their interactions by in vitro assays, cell-based assays, and next we found that JIP-3 is a kinase substrate of ROCK1 by kinase, in vitro kinase assay.

Interviewer – Annalisa VanHookSo, your in vitro experiments showed that ROCK1 binds to JIP-3 and that ROCK1 phosphorylates JIP-3?

Interviewee – Pat OngusahaWe discovered that ROCK1 phosphorylates JIP-3 and we did mass spec on the phosphorylation sites and then we mutate those sites and did the experiment and found that if the cells is expressing the JIP-3 that has mutations on the phosphorylation sites, the ROCK1 phosphorylation sites, it can inhibit UVB-mediated JNK phosphorylation.

Interviewer – Annalisa VanHookSo, the cells require the function of JIP-3 to induce the UV damage signal?

Interviewee – Pat OngusahaYes, in our system, we used keratinocytes to do our experiment and we found that JIP-3 is essential for mediating JNK activation in our cell-based system. And ROCK1 is upstream of JIP-3. ROCK1 phosphorylates JIP-3 and then affects, enhance the recruitment of JNK to JIP-3 and also sensitize the JNK kinase cascade.

Interviewer – Annalisa VanHookThese experiments that we just talked about were done in cultured skin cells, were done in cultured cells. Did you test whether this pathway also functions in the in vivo situation in the animal?

Interviewee – Pat OngusahaYes, we further tested our hypothesis using a mouse model in collaboration with Dr. James Liao in the Brigham and Women’s Hospital, we used ROCK1 heterozygous mice for our study. So, we exposed ROCK1 heterozygous mice and their wild type littermates to UVB. And, interestingly, we found that the epidermis of the ROCK1 heterozygous mice show a lower level of sun damage compared to that of the wild-type littermates. And we also detected a lower level of JNK phosphorylation in the ROCK1 heterozygous mice. So, we believe that from our observation, it strongly suggests that this pathway is conserved in animals.

Interviewer – Annalisa VanHookSo, does it take a certain amount of ultraviolet light to induce the response? I’m guessing that cells that are exposed to just a small amount of ultraviolet light won’t initiate apoptosis.

Interviewee – Pat OngusahaYes, because there is always a balance between the cells, whether the cells want to die or want to repair. So at a lower level of UVB, like physiological level, there will be some apoptotic signal that’s triggered as well as the survival signals. However, I believe that at the low level of UVB there will be more survival signals; hence, the cell will be able to repair its damage. However, with a chronic level of UVB the cells would choose to die because the DNA damage is often irreparable.

Interviewer – Annalisa VanHookWhat sort of practical implications, if any, do you see this research having on treatment or prevention of skin cancer?

Interviewee – Pat OngusahaI think that by identifying ROCK as the sensor of apoptosis will help us to understanding the mechanism of eliminating damaged cells. Because de-regulation of this pathway will result in escape from programmed cell death and that will result in development of skin cancer.

Interviewer – Annalisa VanHookDr. Ongusaha thank you for talking with me.

Interviewee – Pat OngusahaThanks a lot for having me.

Interviewer – Annalisa VanHookDr. Lee, so Pat told me about the research we talked about the experiments, and I will ask you exactly the same question I asked her which was, “What practical implications, if any, do you see this research having on treatment or prevention of skin cancer?”

Interviewee – Sam LeeWell, there has been, just one paper just came out, for the cell, that Rho kinase have play an important role in invading ROCK-mediated tumor metastasis in skin cancer, and we believe that Rho kinase might be an important target to identify certain therapeutic compounds to treat skin cancer and also maybe potential prevention.

Interviewer – Annalisa VanHookDr. Lee thank you for talking with me.

Interviewee – Sam LeeThank you.

Host – Annalisa VanHookPat Ongusaha is lead author and Sam Lee is senior author on a paper published in the November 25th issue of Science Signaling.

music

That wraps up this Science Signaling Podcast. If you have any questions or suggestions, please write to us at sciencesignalingeditors{at}aaas.org. This show is a production of Science Signaling and of AAAS, the Science Society. Our producer is Robert Frederick. I'm Annalisa VanHook, and on behalf of Science Signaling and its publisher, the American Association for the Advancement of Science, thanks for joining us.

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