RxPG News : WNT

Wnt - One Signal, Multiple Pathways: Diversity Comes from the Receptor

Wed, 05 Apr 2006 18:47:37 PST

( from http://www.rxpgnews.com ) Type Wnt into Google Scholar, and you'll get nearly 72,000 hits, revealing the pivotal role this widely conserved family of signaling proteins plays in development and disease. Wnt proteins trigger complex signaling cascades that regulate cell growth, migration, differentiation, and many other aspects of development with the help of numerous interacting components. In the best-understood, canonical pathway, Wnt signaling molecules (called ligands) bind simultaneously to two coreceptors on the cell surface (Frizzled and LRP), allowing β-catenin proteins to stabilize (avoid destruction), enter the nucleus, associate with the transcription factor complex TCF/LEF, and activate genes involved in cell survival, proliferation, or differentiation. Inappropriate activation of β-catenin has been linked to several types of cancer.

Wnt ligands have also been implicated in several alternative, noncanonical pathways, challenging researchers to figure out how proteins that appear so similar at the sequence level can produce such different results. Studies in frogs and zebrafish embryos suggest this diversity derives from engaging multiple pathways, with Wnt5a, for example, triggering an intracellular calcium release that activates calcium-dependent signaling molecules. It's also possible that Wnt5a signals through other receptors (besides the canonical Frizzled receptor) with a Wnt-binding domain, such as the receptor tyrosine kinase-like orphan receptor 2 (Ror2). But because isolating Wnt ligands in a soluble form has proven difficult, scientists have been forced to resort to indirect methods of studying the mechanisms of Wnt studies, which often provided varying and conflicting results.

In a new study, Amanda Mikels and Roel Nusse have developed a technique to purify the Wnt5a protein and directly investigate its contribution to different pathways. They show that soluble Wnt5a proteins can both inhibit and activate the canonical pathway, depending on which combination of receptors is expressed on the cell surface. When Wnt5a interacts with Ror2, the canonical Wnt/β-catenin pathway is inhibited; when it engages Frizzled and LRP, the β-catenin pathway is activated.

The researchers modified a Wnt purification technique previously established in their lab to harvest Wnt5a proteins from cells engineered to overexpress the mouse Wnt5a gene, and confirmed the identity of the protein by examining a key part of its amino acid sequence. Having confirmed the identity of the protein, they compared Wnt5a's capacity to mediate signaling in cells expressing different combinations of the Ror2, Frizzled, and LRP surface receptors. They also examined Wnt5a's capacity to modulate signaling by Wnt3a, which is known to activate the canonical pathway.

First, the researchers tested the possibility that Wnt5a could also activate β-catenin signaling in a cultured cell line (called 293 cells) and found that it could not. But when they treated cells with both Wnt3a and Wnt5a, they discovered that Wnt5a could prevent activation of the β-catenin-dependent TCF transcription factor by Wnt3a. It was initially unclear how this happened. Wnt5a could compete with Wnt3a for the Frizzled receptor, or it might activate a gene that targets β-catenin for destruction. Either way, β-catenin levels should drop following treatment with Wnt5a. Yet β-catenin levels were unaffected; furthermore, Wnt5a didn't interfere with β-catenin's entry into the nucleus. These results indicate that Wnt5a did not block Wnt3a signaling through either of these routes. The researchers also show that Wnt5a doesn't rely on calcium-dependent signals, as had been suggested in previous work. Thus, Wnt5a must act through some other pathway to block β-catenin signaling by canonical Wnts such as Wnt3a.

Previous studies had suggested that Wnt5a might be able to bind another cell-surface receptor, Ror2, based on evidence that blocking expression of either Wnt5a or Ror2 produces the same effects in animals. And this line of investigation proved fruitful: Mikels and Nusse found that Ror2 is needed for Wnt5a-mediated repression of canonical β-catenin signaling. Additionally, by creating multiple Ror2 constructs lacking different combinations of their binding domains, they showed that Wnt5a binding triggers Ror2-mediated signaling inside the cell.

Interestingly, under very specific conditionswhen the coreceptors Frizzled 4 (Frz4) and LRP5 are presentWnt5a can actually trigger β-catenin accumulation and activate canonical β-catenin gene targets. Since 293 cells do not normally express Frz4, but do express Ror2, the predominant signal prompted by Wnt5a in these cells is inhibition of β-catenin signalingindicating that different combinations of cell-surface receptors drive different signaling outcomes for Wnt5a.

Whether Wnt5a inhibits β-catenin signalingperforming its job as a tumor suppressoror activates β-catenin's cell growth and proliferation targetssetting the stage for tumor formationdepends on which receptors are present on the surface of the cell in question. The next challenge will be to identify the mechanism through which Wnt5a blocks the β-catenin pathway. By showing that one Wnt ligand can function through two separate pathways, Mikels and Nusse have opened the floodgates for researching the possibility of dual functionality in the 19 mammalian homologs identified so far. This ability to stimulate different receptors with distinct results is unique for Wnt proteins, but it has been documented in other systems and may well represent an alternate strategy for effecting flexible responses under changing conditions.

Prostate cancer manipulates Wnts signaling proteins in bony metastasis

Tue, 06 Sep 2005 20:39:38 PST

( from http://www.rxpgnews.com ) New research by scientists at the University of Michigan's Comprehensive Cancer Center suggests that prostate cancer manipulates an important group of signaling proteins called Wnts (pronounced wints) to establish itself in bone. By changing the amount and activity of Wnt proteins, prostate cancer cells upset the normal balance between formation and destruction of bony tissue.

There is strong evidence that Wnt proteins play a central role in regulating normal skeletal development in an embryo, says Christopher L. Hall, Ph.D., a senior research fellow in urology at U-M. But this is the first time Wnts have been shown to be involved in abnormal bone production in adult animals with prostate cancer.

Hall is first author of a paper to be published in the Sept. 1 issue of Cancer Research, which presents results from U-M studies of Wnt proteins in human prostate cancer cell lines and in laboratory mice injected with prostate cancer cells.

Normal bone growth and remodeling depends on a controlled balance between production of new bone and resorption of existing bone, says Evan T. Keller, D.V.M., Ph.D., a professor of urology and pathology in the U-M Medical School, who directed the U-M study. When a tumor forms in bone, it upsets this balance.

Several types of cancer metastasize to bone, according to Keller, but most of them tip the balance toward destruction producing what scientists call osteolytic lesions, or holes in the bone. Prostate cancer is unique in its ability to trigger increased bone production, which creates what's called an osteoblastic lesion.

In metastatic prostate cancer, we think that both processes are going on, Keller says. Our hypothesis is that prostate cancer cells first induce more bone resorption to help the invading cells become established in bone. But then there's a switch to increased bone production. Although we don't know the exact mechanism responsible for the switch, we know that it's related to the activity of Wnt proteins in prostate cancer cells.

In the first phase of their research, U-M scientists measured the amount of Wnt protein in cells from normal human prostate tissue, localized prostate cancer and metastatic prostate cancer cells. Using the same cell lines, they also looked for the presence of a protein called DKK-1, which is known to inhibit Wnt activity. They discovered that the amounts of Wnt and DKK-1 protein present in human prostate cells varied inversely with the developmental stage of prostate cancer.

As the cancer progressed, DKK-1 levels went down, Hall says. Cells with osteoblastic activity had high levels of Wnt activity and low levels of DKK-1, while cells with osteolytic activity showed decreased Wnt activity and high levels of DKK-1.

Our results suggest that DKK-1 may act like a switch on prostate cancer cell activity, Keller says. When we altered the cells to increase the amount of active DKK-1, it blocked Wnt's signal, changing prostate cancer cells from an osteoblastic to a highly osteolytic cell line.

To test their hypothesis, U-M scientists injected human prostate cancer cells into the tibias, or long leg bones, of one group of immune-deficient mice. Twelve weeks later, U-M researchers removed and examined bone tumors from the mice. They found that these mice produced tumors with a dense overgrowth of bone. A second group of mice, injected with prostate cancer cells made to express the Wnt inhibitor, DKK-1, developed highly osteolytic tumor lesions, which destroyed most of the bone.

This demonstrated that Wnts promote the overproduction of bone by prostate cancer cells, Keller says.

In previous research, the U-M team found that preventing the osteolytic changes associated with bone resorption also prevented prostate cancer from establishing itself in bone. By learning how DKK-1 blocks Wnt's signal to prostate cancer cells, they hope to learn how to control physical changes in bone that encourage the development of metastatic tumors.

Our goal is to find ways to manipulate this Wnt pathway to slow the growth of tumors in bone or decrease the tumor-associated pain, Keller says. We won't be able to stop the primary tumor from releasing cells, but by preventing early bone resorption, we may be able to prevent metastatic cells from getting a foothold in bone.

In future research, U-M scientists will try to identify which of the nearly 20 known Wnt proteins is involved in bone changes associated with metastatic prostate cancer.

Ryk, Wnts and Frizzled3 receptors in neuronal regeneration - Study

Mon, 15 Aug 2005 20:48:38 PST

( from http://www.rxpgnews.com ) The same family of chemical signals that attracts developing sensory nerves up the spinal cord toward the brain serves to repel motor nerves, sending them in the opposite direction, down the cord and away from the brain, report researchers at the University of Chicago in the September 2005 issue of Nature Neuroscience (available online August 14). The finding may help physicians restore function to people with paralyzing spinal cord injuries.

Growing nerve cells send out axons, long narrow processes that search out and connect with other nerve cells. Axons are tipped with growth cones, bearing specific receptors, which detect chemical signals and then grow toward or away from the source.

In 2003, University of Chicago researchers reported that a gradient of biochemical signals known as the Wnt proteins acted as a guide for sensory nerves. These nerves have a receptor on the tips of their growth cones, known as Frizzled3, which responds to Wnts.

In this paper, the researchers show that the nerves growing in the opposite direction are driven down the cord, away from the brain, under the guidance of a receptor, known as Ryk, with very different tastes. Ryk sees Wnts as repulsive signals.

"This is remarkable example of the efficiency of nature," said Yimin Zou, Ph.D., assistant professor of neurobiology, pharmacology and physiology at the University of Chicago. "The nervous system is using a similar set of chemical signals to regulate axon traffic in both directions along the length of the spinal cord."

It may also prove a boon to clinicians, offering clues about how to grow new connections among neurons to repair or replace damaged nerves. Unlike many other body components, damaged axons in the adult spinal cord cannot adequately repair themselves. An estimated 250,000 people in the United States suffer from permanent spinal cord injuries, with about 11,000 new cases each year.

This study focused on corticospinal neurons, which control voluntary movements and fine-motor skills. These are some of the longest cells in the body. The corticospinal neurons connect to groups of neurons along the length of spinal cord, some of which reach out of the spinal cord. They pass out of the cord between each pair of vertebrae and extend to different parts of the body, for example the hand or foot.

Zou and colleagues studied the guidance system used to assemble this complex network in newborn mice, where corticospinal axon growth is still underway. Before birth, axons grow out from the cell body of a nerve cell in the motor cortex. The axons follow a path back through the brain to the spinal cord.

By the time of birth, the axons are just growing into the cord. During the first week after birth they grow down the cervical and thoracic spinal cord until they reach their proper position, usually after seven to ten days.

From previous studies, Zou and colleagues knew that a gradient of various Wnt proteins, including Wnt4, formed along the spinal cord around the time of birth. Here they show that two other proteins, Wnt1 and Wnt5a are produced at high concentrations at the top of the cord and at consecutively lower levels farther down.

They also found that motor nerves are guided by Wnts through a different receptor, called Ryk, that mediates repulsion by Wnts. Antibodies that blocked the Wnt-Ryk interaction blocked the downward growth of corticospinal axons when injected into the space between the dura and spinal cord in newborn mice.

This knowledge, coupled with emerging stem cell technologies, may provide the most promising current approach to nervous system regeneration. If Wnt proteins could be used to guide transplanted nerve cells -- or someday, embryonic stem cells -- to restore the connections between the body and the brain, "it could revolutionize treatment of patients with paralyzing injuries to these nerves," Zou suggests.

"Although half the battle is acquiring the right cells to repair the nervous system," he said, "the other half is guiding them to their targets where they can make the right connections."

"Understanding how the brain and the spinal cord are connected during embryonic development could give us clues about how to repair damaged connections in adults with traumatic injury or degenerative disorders," Zou added.