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Mutations in Cockayne Syndrome-Associated Genes (Csa and Csb) Predispose to Cisplatin-Induced Hearing Loss in Mice.
Related Articles Mutations in Cockayne Syndrome-Associated Genes (Csa and Csb) Predispose to Cisplatin-Induced Hearing Loss in Mice. J Neurosci. 2016 Apr 27;36(17):4758-70 Authors: Rainey RN, Ng SY, Llamas J, van der Horst GT, Segil N Abstract UNLABELLED: Cisplatin is a common and effective chemotherapeutic agent, yet it often causes permanent hearing loss as a result of sensory hair cell death. The causes of sensitivity to DNA-damaging agents in nondividing cell populations, such as cochlear hair and supporting cells, are poorly understood, as are the specific DNA repair pathways that protect these cells. Nucleotide excision repair (NER) is a conserved and versatile DNA repair pathway for many DNA-distorting lesions, including cisplatin-DNA adducts. Progressive sensorineural hearing loss is observed in a subset of NER-associated DNA repair disorders including Cockayne syndrome and some forms of xeroderma pigmentosum. We investigated whether either of the two overlapping branches that encompass NER, transcription-coupled repair or global genome repair, which are implicated in Cockayne syndrome and xeroderma pigmentosum group C, respectively, modulates cisplatin-induced hearing loss and cell death in the organ of Corti, the auditory sensory epithelium of mammals. We report that cochlear hair cells and supporting cells in transcription-coupled repair-deficient Cockayne syndrome group A (Csa(-/-)) and group B (Csb(-/-)) mice are hypersensitive to cisplatin, in contrast to global genome repair-deficient Xpc(-/-) mice, both in vitro and in vivo We show that sensory hair cells in Csa(-/-) and Csb(-/-) mice fail to remove cisplatin-DNA adducts efficiently in vitro; and unlike Xpc(-/-) mice, Csa(-/-) and Csb(-/-) mice lose hearing and manifest outer hair cell degeneration after systemic cisplatin treatment. Our results demonstrate that Csa and Csb deficiencies predispose to cisplatin-induced hearing loss and hair/supporting cell damage in the mammalian organ of Corti, and emphasize the importance of transcription-coupled DNA repair in the protection against cisplatin ototoxicity. SIGNIFICANCE STATEMENT: The utility of cisplatin in chemotherapy remains limited due to serious side effects, including sensorineural hearing loss. We show that mouse models of Cockayne syndrome, a progeroid disorder resulting from a defect in the transcription-coupled DNA repair (TCR) branch of nucleotide excision repair, are hypersensitive to cisplatin-induced hearing loss and sensory hair cell death in the organ of Corti, the mammalian auditory sensory epithelium. Our work indicates that Csa and Csb, two genes involved in TCR, are preferentially required to protect against cisplatin ototoxicity, relative to global genome repair-specific elements of nucleotide excision repair, and suggests that TCR is a major force maintaining DNA integrity in the cochlea. The Cockayne syndrome mice thus represent a model for testing the contribution of DNA repair mechanisms to cisplatin ototoxicity. PMID: 27122034 [PubMed - indexed for MEDLINE]Read more...
WNT Proteins and Noggin Proteins
From Rockefeller University
Rockefeller scientists identify 'natural' proteins that push stem cells to produce hair, not skin
The clearest picture to date of how two proteins determine the destiny of a stem cell that is genetically programmed to develop into either hair or skin epidermis is emerging with mouse embryos as models for human biology from the Howard Hughes Medical Institute at Rockefeller University. The scientists' latest results are reported in this week's (March 20) issue of the journal Nature.
The proteins, called Wnt and noggin, act in concert to set the stage for the stem cell's developmental pathway into a hair follicle rather than skin, says HHMI investigator Elaine Fuchs, Ph.D., professor and head of the Laboratory of Mammalian Cell Biology and Development at Rockefeller.
These two proteins help change the stem cell's shape so that it can separate from adjoining cells and move downward -- a developmental step that is essential for a hair follicle to form from a stem cell.
Because the Wnt and noggin proteins occur naturally in humans, the research of Fuchs and her research team may enhance understanding of stem cells in humans. "These results might prove to be clinically relevant," Fuchs adds.
The Wnt pathway involved in hair growth has already been implicated in the spread of some cancers, such as colon and breast cancer. In addition, the same process that leads to the separation of a stem cell from other cells may shed insight into how a cancer cell metastasizes, or spreads, from its host tumor, Fuchs explains.
The research may also prove relevant to a much less serious but more common condition, baldness.
"Skin turns over every two weeks, so there is an enormous reservoir of stem cells there," Fuchs notes. "To understand the biology and development of stem cells in general, we are trying to answer the question of whether we can coax some 'skin' stem cells to become hair. These findings reveal some of the natural signals that promote the process of forming hair follicles."
While at the University of Chicago, before joining Rockefeller University in 2002, Fuchs and her research team created an extraordinarily hairy mouse by altering its genes to grow hair follicles out of skin. The hairy mouse demonstrated that the researchers had identified elements of the molecular pathway that leads to hair follicle growth.
The latest study, at Rockefeller University, identifies the external signals that are naturally present in developing skin and that stimulate the production of hair follicles.
Additionally, on a basic science level, the study provides further support to the idea that cell parts known as adherens junctions, once thought useful only as the glue that holds cells of a tissue together actually play an important role in controlling when certain genes are turned on or off, thus transforming the essential nature of the cell.
The study also describes in detail how external protein growth factors produced outside of the stem cells work to activate genetic changes within the cells that prompt hair follicle formation.
"Before this, we didn't know how multiple growth factors collaborated to cause changes within the cell," says the first author, Colin Jamora, Ph.D., a postdoctoral researcher in the Fuchs lab. "Now we know how two of the known ones target a specific gene to change the cell's function."
In the beginning…
In a developing mouse embryo, a sheet of tightly adhering epithelial stem cells form on the body surface. Beginning at embryonic day 13, some of these stem cells receive "growth signals" that tell them to unlink from neighboring stem cells and move downward to form a pocket that will become a hair follicle. Surrounding cells that don't receive these messages continue to develop into the skin cells that form the epidermis, the body's waterproof outer coat. While stem cells at the body surface are forming either skin epidermis or hair, other stem cells in the embryo are differentiating in a similar way, migrating away from that sheet of cells to form teeth, lungs and other organs.
Stem cells that create epidermis or hair have become a model system to study, because they are plentiful in adult skin and they can be maintained in a Petri dish in the laboratory, says Fuchs. The skin epidermis is a multi-layered tissue, and at the innermost or basal layer, stem cells give rise to progeny that divide several times before they are pushed upward and differentiate to produce the body's barrier to keep harmful microbes out and fluids in. The cells that reach the skin surface are dead, and sloughed off, continually replaced by inner layer cells moving outward. "Every two weeks, the epidermis is nearly brand new," she says.
Adult stem cells taken from both humans and mice can be maintained in laboratory culture, and continually propagated. In that way, Fuchs says, researchers can study the genes and proteins involved in turning stem cells into epidermis or hair follicles.
Fuchs and her research team previously discovered that a protein called beta-catenin is a key player in formation of hair. This finding has contributed to the recognition that accumulation of this protein in certain specific cells may be a critical, early step in selecting the developmental pathway of a number of stem cells in the body.
The Rockefeller scientists also found that beta-catenin works in concert with a transcription factor known as Lef-1 (lymphoid enhancer factor). A transcription factor is a protein that can combine with other proteins (in this case, beta-catenin) so that it can turn certain genes in the cell's DNA on or off. The Fuchs lab found that in mice, Lef-1 is expressed (produced) in stem cells that become hair follicles, but not in stem cells that develop into skin epidermis.
In other words, stem cells destined to become hair contain two nuclear proteins -- beta-catenin and Lef-1 -- that are not found in stem cells fated to become skin epidermis. The Rockefeller scientists suspected that beta-catenin and Lef-1 worked together to produce changes in the stem cell that pushed it to "morph" into hair, but they didn't know how, at that time.
Proof of their findings came when the scientists altered genes in experimental mice to over produce beta-catenin and Lef-1. Skin cells on the mice produced luxuriant hair.
However, these same genetic changes form benign tumors around the new hair follicles because the beta-catenin continually pushes new stem cells to form hair. "Such genetic manipulation is obviously not an answer to human hair woes," Fuchs says. v The Rockefeller researchers then searched for the natural triggers that cause both beta-catenin and Lef-1 to form hair without genetic manipulation of the stem cells.
Proteins that cause the cell to change shape
The new research summed up in the Nature paper now paints a more complete picture of the molecular changes involved in hair follicle formation, says Jamora. The Fuchs research team found that proteins that help the cell maintain its shape, collectively called the cytoskeleton, are involved in the decision to change that shape to form hair follicles.
Before stem cells differentiate, they are locked together in tight sheets, zipped to one another. The protein that forms the "teeth" of these zippers is known as E-cadherin; it sticks outside the membrane of each stem cell, and zips together with other E-cadherins in nearby stem cells. E-cadherins are called "adhesion" proteins because they stick like Velcro to each other to help maintain both the shape of the cell and its link to other cells.
"When the process of forming this sheet of stem cells begins, cells touch each other and maintain contact by joining single E-cadherin proteins together on adjacent cells," says Jamora. "This triggers the structural proteins inside the cell to start linking to the actin cytoskeleton. "
That allows the cell to change shape, so that they can zip up tight, locking together through all the many E-cadherin proteins found on the outside of the cell, he says.
Any extra beta-catenin produced within these cells that is not used to link E-cadherin to the actin cytoskeleton is quickly gobbled up by special enzyme "machinery" within the cell body, the researchers say. These stem cells become skin.
Fuchs and her team then clarified what happens when that same cell receives growth signals to change shape and become a hair follicle. After years of research using a series of knockout mice and lab experimentation with their stem cell cultures, the researchers found that both the Wnt and noggin growth factors are needed as simultaneous input to the stem cell.
First, noggin signals the cells to make the Lef-1 transcription factor. Then, the Wnt protein prompts a cascade of signals that turn off the machinery that degrades excess beta-catenin. This allows beta-catenin proteins to build up inside the stem cell. This excess beta-catenin binds to Lef-1.
Once in the nucleus of the stem cell, the beta-catenin/Lef-1 complex reduces the transcription of the gene that produces E-cadherin. By reducing the ongoing synthesis of the E-cadherin protein that is constantly needed to keep cells stuck together, the cell can loosen from others around it. Without as much E-cadherin there to bind to the beta-catenin-actin cytoskeleton complex, the structure of the cell changes, allowing it to migrate down between the other stem cells, Fuchs says.
Using mice genetically altered not to produce noggin, the researchers showed that the Lef-1 transcription factor was not being produced. Experiments in which the level of E-cadherin was kept high blocked production of hair follicles, because E-cadherin production must be reduced in order for stem cells to loosen and reorganize to form follicles. Together these experiments verified the importance of the beta-catenin/Lef-1 pathway in hair follicle formation.
The finding that the beta-catenin/Lef-1 transcription complex turns down the expression of the gene that makes E-cadherin is completely novel, says Jamora, since this complex was only known to turn genes on.
Hair, cancer, and more
The description of how Wnt and noggin produce structural changes in a stem cell may ultimately shed light on several developmental and disease processes, Fuchs says. Mutations in E-cadherin and problems in the Wnt signaling pathway have already been linked to some cancers, says Fuchs. "The reason why tumor cells don't interact properly with other cells may be that their levels of adherens junction proteins are not maintained," she says. "For example, squamous cell skin cancers are large masses of cells that invaginate downward."
Too much, or too little, E-cadherin is "a bad thing," Fuchs says. With too much, hair can't develop. With too little, cancer may result.
The study was funded by a grant from the National Institutes of Health.
Founded by John D. Rockefeller in 1901, The Rockefeller University was this nation's first biomedical research university. Today it is internationally renowned for research and graduate education in the biomedical sciences, chemistry, bioinformatics and physics. A total of 22 scientists associated with the university have received the Nobel Prize in medicine and physiology or chemistry, 18 Rockefeller scientists have received Lasker Awards, have been named MacArthur Fellows, and 11 have garnered the National Medical of Science. More than a third of the current faculty are elected members of the National Academy of Sciences.