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UCSD RESEARCH DESCRIBES FUNCTION OF ENZYME INVOLVED IN WIDE RANGING BIOLOGICAL PROCESSES
Work is featured on the cover of Science

Researchers at University of California, San Diego (UCSD) School of Medicine have found that an important enzyme previously thought to be associated only with inflammation is also a key factor in skin formation and in programmed cell death.

Their findings, reported in two papers and featured on the cover of the April 9 issue of Science, have important implications for the pharmaceutical industry and set the stage for future development of therapies in three very different areas - skin diseases, cancer and inflammatory diseases such as asthma, rheumatoid arthritis and septic shock.  This issue of Science carries two other papers and an accompanying editorial regarding additional insights into this enzyme's activity.

"These papers provide the basic understanding that is essential for the rational development of very specific drugs," said Michael Karin, Ph.D., professor of pharmacology and director of the Cancer Biology Program at the UCSD Cancer Center, who directed the UCSD research team. Karin is also an American Cancer Society Research Professor.

The enzyme, a protein kinase complex called I-kappa-B kinase (or IKK), and its three sub-units - named alpha, beta and gamma -- were first identified in late 1996 in Karin's lab.  (Protein kinases are enzymes that regulate the function and metabolism of other proteins by adding phosphate groups to them.)  Through that landmark work, and work done elsewhere, it was shown that IKK is responsible for controlling the body's inflammatory response, which is the first line of defense from microbial infections.  Because the alpha and beta sub-units of IKK are both protein kinases that are similar in their chemical structure and are usually seen bound together, researchers believed they performed the same function.

In this newest work, Karin and colleagues tested that theory by developing cell lines that lack the activity of either the alpha or beta sub-units.  They found that the IKK-beta sub-unit is essential for activation of the entire IKK complex by responding to chemical signals that trigger the inflammatory response.  In contrast, when the IKK-alpha sub-unit was inactivated, there was no change in the capacity of the cells to respond to the signals.

To further test the in vitro results, the researchers genetically engineered mice lacking the gene necessary to produce IKK-alpha.  They found that these mice were able to mount normal inflammatory responses, confirming the importance of IKK-beta in this process.  (Karin's group has also completed work with mice lacking IKK-beta, but they have not yet reported their results.)

However, the IKK-alpha deficient mouse model revealed, to the surprise of the researchers, that IKK-alpha serves a completely unimagined function: It is responsible for a key step in the formation of the upper layer of skin, called the epidermis.

Specifically, IKK-alpha controls the proliferation and differentiation of basal cells, which lie in the base of the epidermis.  Normally, as they mature, basal cells work their way up through the epidermis and ultimately form the thin layer of keratinized cells on the skin surface that serve as a barrier, preventing the loss of fluids and easy entry of microorganisms.   The absence of IKK-alpha results in uncontrolled production of basal cells, and complete absence of the uppermost layer of keratinized skin cells.

"This finding is important because basal cell carcinoma -- a common form of skin cancer -- is due to uncontrolled proliferation of basal cells," Karin said. "It's very likely that some of the things that go wrong in that cancer are directly related to the new pathway we've identified."

In parallel work that may have broader implications, the researchers confirmed that IKK-beta is a master controller of inflammatory responses and is also involved in protecting cells from programmed cell death, a process known as apoptosis.

Normally, the inflammatory response is the body's protective response against microbial invaders. In some cases, however, the response can be overly aggressive and damage healthy tissue; in extreme situations death can result. Inflammation can also become chronic, leading to diseases such as asthma or arthritis.

Researchers have known that IKK and a protein called nuclear factor-kappa-B (NF-kappa-B) are central players in the cascade of events leading to the inflammatory response.  NF-kappa-B is a master transcription factor - a protein involved in gene regulation. IKK regulates activity of NF-kappa-B, but exactly how this occurs has not been known until now.

In both cell cultures and in the IKK-alpha deficient mice, the UCSD team showed that as long as IKK-beta is active, the inflammatory response is normal.  On the other hand, inactivation of IKK-beta hindered the inflammatory response.

"Prior to this work it was thought you would have to inhibit both of IKK's sub-units, alpha and beta, to block activation of NF-kappa-B and the inflammatory response.  Now it is clear that the pharmaceutical industry should focus its efforts on developing inhibitors only for IKK-beta," Karin said.

Previous work has also shown that certain tumors that did not respond well to either radiation or chemotherapy contained high levels of NF-kappa-B.  Subsequent work confirmed that NF-kappa-B actively protects cells against cell death.

"Because we now know that the IKK-beta sub-unit is responsible for activation of NF-kappa-B, which protects cells from this killing effect, it stands to reason that inhibitors for IKK-beta should sensitize tumors to conventional chemotherapy or radiation," Karin said.   "But we must learn how to selectively inhibit the beta enzyme in the tumor and not in other cell types.  This will be a challenge, but I believe a drug with the right properties will be found."

A molecular biologist, Karin ranks 12th on the list of "hottest biomedical researchers of the 1990s" by Science Watch, a publication that tracks trends and performance in basic research.   Since 1981, more than 70 of his papers have been cited over 100 times each, and five papers have been cited more than 1,000 times each.

Co-authors on the paper titled "Abnormal Morphogenesis But Intact IKK Activation in Mice Lacking the IKK-alpha Subunit of I-kappa-B Kinase" are Yinling Hu, Veronique Baud and Mireille Delhase, Department of Pharmacology; Peilin Zhang, Department of Pathology; Thomas Deerinck and Mark Ellisman, Department of Neurosciences; Randall Johnson, Department of Biology.  Co-authors on the paper titled "Positive and Negative Regulation of I-kappa-B Kinase Activity Through IKK-beta Subunit Phosphorylation" are Delhase, Makio Hayakawa and Yi Chen, Department of Pharmacology.  Other co-workers involved in the generation and analysis of IKK-beta-deficient mice are Zhi-Wei Li, Wenming Chu and David Rothwarf. The research was supported by the National Institutes of Health (National Institute of Environmental Health Sciences, National Institute of Allergy and Infectious Diseases), the American Cancer Society and the U.S. Department of Energy.

Note: Copies of the papers may be obtained from Gabriel Paal at the AAAS News & Information Office, 202/326-6421 or email gpaal@aaas.org.