Famous Factor Triggers Tanning Response

New evidence from HMS researchers brings to light a novel role for the transcription factor p53, famous for its tumor-suppressing capabilities. This oncological celebrity was found to be part of the tanning response to UV light, according to findings published in the March 9 Cell. Though p53 upregulation following UV stimulation has been known for about 25 years, its role in skin pigmentation has been left unexplored.

Image adapted from original courtesy of David Fisher

Shedding light on p53. UV causes p53 to release melanocyte-stimulating hormone (MSH), which induces melanin synthesis in skin (upper right). The skin of mice lacking p53 did not show melanin accumulation after UV exposure (lower right).

David Fisher, HMS professor of pediatrics at the Dana–Farber Cancer Institute, previously found that UV light induces pigment-producing melanocyte-stimulating hormone (MSH) in keratinocytes. The finding, reported in the Sept. 21, 2006, Nature, overturned the belief that UV induces pigmentation by acting directly on melanocytes rather than keratinocytes (see Focus, Oct. 13, 2006).

Yet the researchers had not uncovered the molecular connection between UV stimulation and MSH production. To determine the players involved, the Dana–Farber team scrutinized the POMC gene, from which MSH is derived, looking for a regulatory DNA element common to the human, rat, and mouse that would identify the transcription factor linking UV stimulation with subsequent MSH release. They noticed the sequence for p53 and thought, “Of course!” said Fisher, of this protein commonly mutated in cancer. The tumor suppressor has long been known to respond to DNA damage, but a connection to the common form of DNA damage induced by sunlight had not been studied in this context. Normal p53 function includes activating genes that stop cell growth and induce apoptosis.

Fisher and his team exposed human and mouse keratinocytes to UV light and found that p53 increased three hours before MSH increased, suggesting that p53 signals the activation of MSH. “The timing fit,” Fisher said. Overexpression of p53 also led to an MSH surge.

While it appeared that p53 was sufficient to trigger MSH, it was unclear whether the protein was necessary to induce the hormone’s release. To determine this, the researchers used a variety of inhibitors to block p53 expression. They found that with the protein blocked, MSH did not increase in response to UV light. And the ears and tails of p53-lacking mice did not tan when exposed to UV light, whereas the ears and tails of the p53-possessing wild types turned dark brown.

MSH is cleaved from the large, multicomponent gene POMC, which codes for a variety of biologically active peptides. Of note to Fisher, UV-responsive beta-endorphin resides among POMC’s 241 amino acid residues. Because p53 stimulates beta-endorphin, Fisher speculates that this endogenous opiate might be responsible for pain relief following sunburn and the addictive qualities of tanning that promote sun-seeking behavior.

—Molly McElroy

Assay Identifies Molecular Saboteurs of Dengue Virus

Certain protein kinase inhibitors, now in clinical development against cancer, unexpectedly have been found to block the dengue virus, for which no pharmacological treatment exists. Using immunofluorescent imaging techniques, HMS researchers discovered that inhibitors of c-Src protein kinases prevented the assembly of dengue virus particles in primate and mosquito cells. The findings were reported in the Feb. 27 Proceedings of the National Academy of Sciences.

Transmitted by mosquitoes and related to the West Nile virus, dengue virus can cause head and joint aches symptomatic of dengue fever. This condition sometimes progresses to the deadly dengue hemorrhagic fever, or dengue shock syndrome. A report last year indicated that dengue virus, whose mechanism of infecting cells remains unclear, annually afflicts 50 to 100 million people with dengue fever and causes 250,000 to 500,000 cases of the more severe shock syndrome.

With expanding mosquito habitats and increasing cases of the severe form of dengue fever, Priscilla Yang, HMS assistant professor of microbiology and molecular genetics, and postdoctoral researcher Justin Chu wondered whether molecules directed at cellular targets could be used to combat the virus.

“We wanted to pick a third-world disease, a less studied virus,” Yang said. To have a head start on finding a treatment, the researchers used drugs already in development.

“There are lots of molecules in clinical development as kinase inhibitors,” Yang said. “They are known to be orally bioavailable, but not to be toxic.”

But which kinase inhibitor to choose? To whittle down the possibilities, Yang and Chu first developed an immunofluorescent imaging assay to detect the amount of dengue infection in cells incubated with the virus. The assay measured dengue-virus envelope protein, a viral-encapsulating molecule. Three days after cotreatment with the virus and mycophenolic acid (an immunosuppressive agent that is known to also inhibit dengue virus in vitro), the number of positively stained cells showed a dose-dependent decrease in the viral envelope protein. Satisfied that their assay could detect inhibition of dengue, the researchers went on to co-incubate with the virus non-cytotoxic concentrations of 120 different kinase inhibitors.

“We found several hits, including Src kinases,” Yang said. The c-Src protein kinase inhibitors dasatinib and AZD0530 showed particularly strong inhibitory effects on the virus. Yang and Chu went on to demonstrate that silencing c-Src protein prevented the replication of the dengue virus in human hepatoma cells and that the inhibitors’ virus-blocking abilities were due to their inhibition of viral-particle assembly. The results indicate that host-cell factors such as protein kinases are viable antiviral drug targets.

Because dengue virus belongs to the Flavivirus genus, which includes West Nile virus and tick-borne encephalitis, it is possible that Src kinase treatment could be effective against other types of viral infection.

—Molly McElroy

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