Molecule Orientated Discovery: Driving Wound Recovery

INTERVIEW WITH:

Takehiko Yokomizo
Professor of Biochemistry
Juntendo University Graduate School of Medicine

People get wounds for various reasons. In addition to injury from falls and burns, wounds to organs such as ulcers of the digestive tract are common. However, many mysteries still remain in the wound recovery process. Steady progress is being made by research into pharmaceuticals that promote recovery. We interviewed Prof. Takehiko Yokomizo at the Biochemistry department of Juntendo University, Tokyo, who is working on receptors for lipid mediators, such as leukotrienes, and their relationship with wound recovery.

“In my laboratory, we investigate anything on the theme of G protein-coupled receptors (GPCR). While we have conducted much research on lipid mediators with actions vital to wound recovery, such as leukotriene (LT), 12-hydroxyheptadecatrienoic acid (12-HHT) and their receptors, we also conduct research into receptors that bind other molecules as ligands. To be honest, I didn’t originally have a particularly strong interest in wound recovery. Instead, I arrived at wound recovery in the course of researching the BLT2 receptor. My research is always ‘molecule-oriented’.”

Molecule-Oriented Research

Spurred in part by the recommendation of a professor when working as an obstetrician and gynecologist, Prof. Yokomizo started research under Prof. Takao Shimizu, a leading expert in prostaglandin research. Initially his research focused on lipid metabolizing enzymes and then progressed to the mechanism of action of bioactive lipids. He then decided to challenge himself to do research into receptors during the preparation period for his overseas study. Since the research was to be done in such a short time before his overseas study, he attempted cloning of BLT1 cDNA using an unconventional method, a cDNA subtraction, and as a result, he discovered the gene for the leukotriene B4 (LTB4) receptor(1). LTB4 had been known to act as a potent chemotactic factor for neutrophils, eosinophils, and macrophages, but nobody had succeeded in identifying its receptor until his discovery. During a bacterial infection, neutrophils usually gather at the site of infection (known as swarming). Knocking out BLT1 gene eliminates the awareness of neutrophils for bacterial infection, reducing anti-bacterial efficiency due to the lack of swarming and increasing susceptibility to such infections. Additionally, inflammation of the bronchi in bronchial asthma is mainly due to eosinophils. Knocking out BLT1 greatly reduces their migration, resulting in suppression of bronchial inflammation lessening the asthmatic response. “Among white blood cells, it is the neutrophils that attack bacteria, while eosinophils mainly attack parasites. Bacterial infection seems to have been a grave threat to organisms throughout the long history of evolution, and there exist several other receptors besides BLT1 that induce neutrophil chemotaxis. This means that bacterial infection is not fatal even if BLT1 is knocked out. In contrast, eosinophils do not appear to have a backup for BLT1, and eosinophil infiltration is completely gone in BLT1-knockout mice infected with parasites. In other words, while LTB4 was well known as a chemotactic factor for neutrophils, in actuality, it has a more important role for eosinophils. These findings first become evident from analysis of isolated molecules using gene-deletion mice.”

Discovery of BLT2, a BLT1 Like Receptor

When analyzing the transcriptional regulation of BLT1 to understand the molecular mechanism behind the  leukocyte-specific expression of the BLT1 receptor, Prof. Yokomizo found a protein coding region for another receptor upstream of the BLT1 locus. The amino acid sequence of the receptor encoded by this gene was 45% similar to that of BLT1, making it a ‘BLT1 like molecule,’ however its function was unknown. After testing more than 30 ligands against this receptor, it was found that it recognized LTB4, and thus was named BLT2(2,3).

 While this BLT2 receptor recognized LTB4 at high concentrations, its affinity for LTB4 was low, to the extent that in vivo LTB4 concentrations did not cause activation. Consequently, Prof. Yokomizo anticipated the existence of another ligand for BLT2 receptor.  After partial purification of BLT2-active lipid from lipid extracts from various organs, an oxidized fatty acid called 12-HHT, belonging to the prostaglandin production pathway, was found to activate BLT2 (4). 12-HHT was known to be produced in large amounts when platelets were activated, such as during wound recovery; however, its physiological action was unknown.  By establishing BLT2 antibodies and staining mouse and human skin, it was found that BLT2 was specifically expressed in keratinocytes in the skin. The production of 12-HHT was also known to be inhibited by administration with aspirin. Prof. Yokomizo and his colleagues, suspecting that BLT2 and 12-HHT were related to wound healing as shown below, conducted further experiments.

“I heard that dermatology physicians knew empirically that aspirin and ibuprofen make wound recovery more difficult, and struck on the idea that perhaps this receptor participated in keratinocyte migration. While BLT1 and BLT2 are expressed in different cells, I knew that they both activate the intracellular signals that move cells, which is why I thought perhaps BLT2 is a receptor for moving keratinocytes. So I decided to observe wound healing in the skin of BLT2 knockout mice.”

Mice recover from wounds quickly, with wounds recovering 50% of their surface area in around three days and being mostly healed in a week. In the wound recovery process, a platelet-containing scab is formed first, then substances for healing the wound are released from the scab. Fibroblasts in the skin reduce the size of the opening to the wound to form a seal while keratinocytes simultaneously migrate inwards from the outside to cover the surface. Knocking out the BLT2 receptor did not lead to any change in the process of fibroblasts sealing the opening to the wound; however, it did slow the rate at which keratinocytes covered the surface. Thus, to verify this process in vitro, a scratch wound migration assay was conducted (see Figure 2).

Scratch wound assays performed using skin keratinocytes from newborn wild-type and BLT2-knockout mice, showed that both cell types recovered from injury at the same, slow, pace in the absence of 12-HHT. Addition of 12-HHT had no effect on the recovery rate for BLT2-deleted mouse cells, however, wound healing was accelerated for the wild-type mice cells, with wound recovery occurring in half the time. These results were the foundation of the discovery that 12-HHT/BLT2 pathway promote migration in keratinocytes. However, the process of arriving at this conclusion was plagued with adversity.

“The wound in the scratch assay would become jagged, or the cells would peel off, which made the establishment of uniform conditions exceptionally difficult. However, introducing an IncuCyte® live-cell analysis system and 96-well WoundMaker™ tool made it possible to make very clean scratches in an instant, and we were able to easily obtain high-precision data with very small error bars. The small size of these error bars surprises a lot of people. A paper summarizing the results obtained using this IncuCyte with mice was published in a major journal(5). It is very convenient to be able to obtain both still images and videos, as well as live kinetic analysis data entirely automatically. ” (See Figure 3, Video 1)

The identified wound recovery-related substances released from scabs include TGF-β, which promotes proliferation and activation of dermal fibroblasts to narrow the wound opening, and VEGF, which promotes angiogenesis to supply the wounded region with nutrients and oxygen. However, the substance that participates in the crucial migration of keratinocytes to cover the surface of the wound had not been previously elucidated. Prof. Yokomizo’s research proved that the lipid-soluble ligand, 12-HHT, is released from the scab and promotes the migration of keratinocytes to cover the wound(5).

The Desire to Produce Research Results that Benefit People

At present, Prof. Yokomizo’s laboratory is working to advance drug discovery research for diabetic skin ulcers and bedsores through the practical application of these research results. Because the openings of bedsores and diabetic ulcers are always exposed, they present considerable problems, such as a greatly increased risk of bacterial infection and reduced quality of life (QOL) for patients.

“One pharmaceutical company discovered a compound that activates BLT2 similarly to 12-HHT. Application of ointments containing this compound and testing them in mice, the rate of wound closure was accelerated. On observing the skin tissues, we obtained data that only the keratinocytes were being activated, with no influence on the process of closing the wound by the fibroblasts. The compound also promoted the migration of keratinocytes in human skin biopsy samples. Presently, in cooperation with the pharmaceutical firm, we are attempting to alter the substance to increase its affinity above that of the original compound and have it adopted clinically as a drug for curing bedsores and ulcers.

I first discovered BLT2 in the year 2000, and the intervening 16 years have passed quickly. While I have consistently maintained a basic stance that ‘I research out of interest’ and ‘there is a thrill to shedding light on something that was a mystery,’ because I am a medical school-affiliated researcher, I feel that I want to engage in research that benefits people, where the chance arises. I feel that maybe BLT2 is a molecule that has the potential for that. I will feel very fortunate if drug development derived from the molecule we found becomes implemented.”

The Essence of Biochemistry

Prof. Yokomizo commands a view over both the entrance to and the exit of biochemical research. While he humbly describes this as “luck,” he was asked if he was given the opportunity to research whatever he wished to, what kind of challenge would he prefer?

“Even now, I think that molecular-oriented research is the essence of biochemistry. First, seize on a molecule that no one else has yet to focus on, and elucidate its function. A molecule that you discover yourself is something like your child. You know more about that molecule than anyone else, and that builds a motivation to continue researching. It is said that there are 900–1000 GPCRs genes in the human genome, and we focus research on six or seven of these receptors. I would like to identify the ligands and clarify the role of a few of these receptors as per BLT1 and BLT2. From the beginning, I liked such experiments that dig deep into individual molecules; however, if possible, I would like to try research that deals with all the GPCRs. For example, making knockout mice of all 1000 types of GPCR and comparing the phenotypes in one experimental system. I suspect this would reveal even-more-important receptors involved in wound recovery. There are many GPCR researchers across the globe, and they publish various papers related to each of the receptors, but their experimental conditions and environments differ, making it difficult to compare them side-by-side. To fairly determine the involvement of each molecule, it is important that they all be assessed in the same arena. Doing something like that is a dream of mine.”

References

1. A G-protein coupled receptor for leukotriene B4 that mediates chemotaxis. Nature (1997) 387, p620-624
2. A second leukotriene B4 receptor, BLT2: a new therapeutic target in inflammation and immunological disorders. J. Exp. Med. (2000) 192, p421-432
3. Cell-specific transcriptional regulation of human leukotriene B4 receptor gene. J. Exp. Med. (2000) 192, p413-420
4. 12(S)-Hydroxyheptadeca-5Z, 8E, 10E-trienoic acid is a natural ligand for leukotriene B4 receptor 2. J. Exp. Med. (2008) 205, p759-766
5. 12-hydroxyheptadecatrienoic acid promotes epidermal wound healing by accelerating keratinocyte migration via the BLT2 receptor. J. Exp. Med. (2014) 211, p1063-1078

This is a translation of an article written by KEYSTONE, co. ltd. and used with permission.