Molecular Middleman Puts Thyroid Hormone in Developmental Signaling Pathway

Photo by Steve Gilbert Photo courtesy of Monica Dentice “One area that will benefit from this work is brain development,” said Antonio Bianco, shown with collaborator Monica Dentice. With colleagues, they discovered a protein involved in activation of thyroid hormone that is regulated by the hedgehog family of developmental signaling proteins.

To many researchers, the thyroid evokes all the drama of a quiet summer day. The butterfly-shaped gland is, in a sense, designed to be uneventful. Its job is to help keep the body’s metabolism on an even keel. It does this by releasing into the bloodstream a hormone that when activated, can travel into the nucleus of cells, turning on and off genes involved in the production of energy. Endocrinologists have known for some time that the active form of thyroid hormone, T3, is regulated by a delicate feedback mechanism so its level in the bloodstream rarely flutters—a fact that may contribute to the thyroid’s laid-back reputation.

“People wonder how a hormone whose serum concentration almost never changes can regulate important physiological pathways,” said Antonio Bianco, HMS associate professor of medicine at Brigham and Women’s Hospital. “They just do not give much credit to thyroid hormone because they are looking in the wrong place, they are looking at the plasma.”

He and his colleagues have been looking at what happens to thyroid hormone once it gets inside cells. What they have found suggests a more exciting picture, one that could make scientists in far-flung fields sit up and take notice. In the June 19 online Nature Cell Biology, they report finding a molecule, WSB1, that regulates how much thyroid hormone is activated. What’s more, WSB1-controlled thyroid hormone activation plays a role not just in homeostasis, but also in a critical developmental pathway. Specifically, the WSB1 findings could shed light on how members of the well-known hedgehog family tell immature cells when to keep proliferating.

Calm Plasma, Stormy Tissue
While levels of T3 in the blood tend to remain stable, they can vary wildly within cells. Tissues such as muscle and brain convert the inactive form of thyroid hormone, T4, into T3 on an as-needed basis—and they do so quickly. In the 1980s, researchers discovered that this conversion is accomplished by a set of enzymes, the deiodinases, that essentially pluck an iodine atom off T4.

Several years ago, Bianco and colleagues discovered that the most important activating deiodinase, D2, is regulated by the ubiquitination pathway. Almost any protein can be ubiquitinated, or tagged for destruction, but it requires a ligase, a molecular adapter that fits the ubiquitination machinery to the protein. The researchers wanted to find the D2 ligase.

Image courtesy of Antonio Bianco, adapted by Rachel Eastwood

Muscle and brain cells convert the inactive form of thyroid hormone, T4, into T3 by means of the enzyme D2 (red). D2 is regulated, in turn, by WSB1 (pink), which allows the ubiquitination machinery (gray) to attach to D2 and tag it for destruction.

They performed a yeast two-hybrid screen, netting about a thousand proteins. The most promising was an odd-shaped molecule called WSB1. “I was not enthusiastic about it, the preliminary data were not encouraging,” Bianco said. Monica Dentice, a newly arrived postdoctoral fellow from Italy, plunged into the project. She found WSB1 interacted with D2. More importantly, it was required for D2 ubiquitination. Dentice and colleagues went on to identify an 18-amino-acid loop on D2 that serves as the WSB1 recognition site. WSB1 was clearly the D2 ligase.

Hedgehog Power
It would turn out to be an even more interesting character. One of the few things known about WSB1 is that it is induced by the hedgehog proteins, a family well studied by Cliff Tabin, HMS professor of genetics. Development is a balancing act between proliferation and differentiation and the hedgehog family is known for accomplishing this feat. Yet there are gaps in understanding the process. For example, one family member, Indian hedgehog (Ihh), promotes proliferation in the developing bone of very young embryos by turning on a hormone, PTHrP, but it was unclear how exactly—by what downstream pathway—it does that.

The researchers thought WSB1 regulation of D2 and thyroid hormone activation might be playing a role. Working with Amitabha Bandyopadhyay and other members of Tabin’s lab, Dentice infected developing chick legs with a virus that overproduces Ihh to see whether D2 levels would decrease in response to hedgehog’s command. They removed the leg a few days later. D2 levels were down and WSB1 was up. They then removed several chick tibias and incubated them with a more readily available hedgehog member, Sonic hedgehog, for 24, 48, and 72 hours. The results were the same—WSB1 expression was up, D2 was down. As expected, PTHrP was elevated.

“So induction of WSB1 and ubiquitination of D2 are part of this pathway,” Bianco said. “Until now details of the connection between Ihh and PTHrP were missing.” He believes that one factor contributing to PTHrP expression is the turning down of D2 and local T3 production, essentially creating a condition of local hypothyroidism. Still, there are missing puzzle pieces. The researchers do not know, for example, how low levels of T3 result in the turning on of PTHrP. In addition, they carried out their experiments on embryonic chicks. “We are just learning about what is regulating WSB1 in the adult,” Bianco said. “We have cloned the promoter of WSB1 and are looking at what is regulating it. That should have a major impact.”

Meanwhile, Bianco believes there is much to be gained just by focusing on the original discovery, namely that WSB1 is the D2 ubiquitin ligase. He is especially excited about the potential therapeutic applications. “This is a very rich subject,” he said. “From a pharmacological point of view, it is self-evident how we can play with this system. If we can somehow interfere with the binding between WSB1 and D2—and we can because we have the 18-amino-acid loop of D2—and if we can produce a molecule that interferes with this binding, we can stop D2 ubiquitination and increase intracellular T3 production without affecting serum T3 concentration.” Such an approach could be used to raise intracellular T3 levels in hypothyroid patients on a tissue-specific basis.

Given the wealth of opportunity raised by the discovery of WSB1’s role in thyroid hormone activation, the hedgehog connection might seem an unexpected bonus. “From the thyroid perspective, we knew the discovery of WSB1 was a very important finding,” he said. “It became so much more exciting when we found that WSB1 is regulated by the hedgehog family.”

—Misia Landau

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