By Dean A. Haycock
Special to BioWorld Today
Some very sickly mice are pointing the way toward a promising new drug target for treating a variety of autoimmune and inflammatory diseases.
The mice appear normal at birth, but soon after their skin becomes inflamed and their bone marrow cells increase in number. They also lose weight, develop severe arthritis in their paws and show signs of autoimmune disease.
"This syndrome looks very much like what happens to mice when they are treated with TNF-a [tumor necrosis factor-alpha], either by injection of the substance or by a transgenic approach," said Perry Blackshear, of the National Institute of Environmental Health Sciences, in Research Triangle Park, N.C., and Duke University Medical Center, in Durham, NC.
TNF-a is a well-studied protein that plays a central role in mediating inflammatory responses, both chronic and acute, in mammals.
Why would these mice overproduce TNF-a? The answer is in their genes. Or, more accurately, it lies in a gene the mice lack. It is explained in a paper appearing in today's Science, dated Aug. 14, 1998, and titled "Feedback inhibition of macrophage tumor necrosis factor-a production by tristetraprolin." The paper was written by Blackshear and his research associates, Ester Carballo and Wi Lai.
The team genetically modified the mice so they could not produce a protein called tristetraprolin (TTP). It is the lack of TTP that appears to be responsible for excess production of TNF-a and the resulting complex syndrome that affects the mice. TTP, under normal conditions, appears to keep TNF-a in check. Without its controlling influence, TNF-a is overproduced.
"Although we could not measure TNF-a in the blood of these animals, we hypothesized that this syndrome was the result of excess TNF-a. To test that, we injected these animals with anti-TNF-a antibodies which are specific for the mouse form and were able to prevent essentially all aspects of the syndrome from developing," Blackshear explained.
The fact that antibodies to TNF-a eliminated the varied symptoms of inflammatory disease in the mice indicates they were producing excess amounts of the protein.
The researchers confirmed that the syndrome resulted from TNF-a production by isolating macrophages from the TTP-deficient mice and showing they overproduced TNF-a relative to controls. The same overproduction was observed in macrophages obtained from the fetal liver of the modified mice. These cells had never been exposed to an environment rich in inflammatory agents like that experienced by older animals.
There are, Blackshear noted, only so many ways the mice could overproduce TNF-a: by increased transcription of the TNF-a gene; by increased translation of its messenger RNA; or by stabilization of the messenger RNA prior to actual synthesis of the TNF-a protein.
"We determined it was through stabilization of the messenger RNA encoding TNF-a," Blackshear said.
TTP is a "zinc finger" protein, one of a large class of proteins that have a characteristic structural feature in which certain amino acids are coordinated by a single atom of zinc.
The zinc finger region of these proteins has been shown in other cases to bind to DNA, RNA, other proteins and non-protein substances. TTP appears to regulate the production of TNF-a by incapacitating the RNA that encodes it.
Blackshear and his co-workers showed TTP binds directly to regions of TNF-a messenger RNA and destabilizes it.
The same agents that stimulate TNF-a stimulate TTP. Even TNF-a stimulates TTP production. In so doing, TNF-a appears to contribute to its own regulation by means of a negative feedback system in which a stimulus evokes an inflammatory response that includes the production of TNF-a and TTP.
"TNF-a is unusual in that it stimulates its own production rather than feedback inhibiting it," Blackshear said.
TNF-a by itself, therefore, appears to positively feed back its own synthetic machinery and so increases to dangerous levels if unregulated. TTP, produced along with TNF-a, keeps TNF-a levels from soaring as they do in the genetically altered mice used in these studies.
"Since TNF-a stimulates TTP production, that permits a novel mechanism by which TTP in turn can feed back to inhibit TNF-a," Blackshear said.
Antibodies to TNF-a and receptors that bind the protein have been proposed as treatments for some diseases caused by uncontrolled inflammatory responses. They have even shown some positive effects in humans.
Sepsis Among Potential Diseases For Treatment
Now, TTP emerges as a promising potential drug target for treating a number of disorders, including septic shock, graft-vs.-host disease, rheumatoid arthritis, Crohn's disease and the physical wasting (cachexia) that accompanies many cancers and AIDS.
"We know now that TTP stimulates TNF-a messenger RNA degradation and thus inhibits TNF-a production. That is a desirable thing to do with a drug in many different diseases. An agent that mimics TTP's effect on that message or potentiates the effect of TTP could be potentially, at least, a useful drug," Blackshear said.
Other possibilities, he added, include stimulating the production of TTP or introducing TTP by gene therapy.
Blackshear's group is negotiating with one company on a collaborative research and development agreement. The negotiations may be completed during the next month.
At the level of basic research, their experiments will be directed at figuring out the molecular mechanisms by which TTP works and investigating the effects of TTP on other RNA messages containing destabilizing elements similar to those present in the TNF-a system.
Blackshear also is interested in studying other members of the class of zinc finger proteins, which includes TTP. *