Embryology Study Offers Clues to Birth Defects

Gregg Duester, Ph.D., professor in the Development and Aging Program at Burnham, Xianling Zhao, Ph.D., and colleagues have clarified the role that retinoic acid plays in limb development.

The study showed that retinoic acid controls the development (or budding) of forelimbs, but not hindlimbs, and that retinoic acid is not responsible for patterning (or differentiation of the parts) of limbs. This research corrects longstanding misconceptions about limb development and provides new insights into congenital limb defects. The study was published online in the journal Current Biology on May 21.

“For decades, it was thought that retinoic acid controlled limb patterning, such as defining the thumb as being different from the little finger,” says Dr. Duester. “However, we have demonstrated in mice that retinoic acid is not required for limb patterning but rather is necessary to initiate the limb budding process.”

By providing a more complete understanding of the molecular mechanisms involved in normal limb development, these findings may lead to new therapeutic or preventative measures to combat congenital limb defects, such as Holt-Oram syndrome, a birth defect characterized by upper limb and heart defects.

New Insights into Limb Formation

Drs. Kazu Matsumoto and Yu Yamaguchi, investigators at Burnham and the University of Connecticut Health Center (U.C.H.C.) have gained new understanding of the role hyaluronan (also known as hyaluronic acid or HA) plays in skeletal growth, cartilage maturation and joint formation in developing limbs.

Significantly, these discoveries were made using a novel mouse model in which the production of hyaluronan is blocked in specific tissues. The Yamaguchi laboratory genetically modified the Has2 gene, which is a critical enzyme for hyaluronan synthesis, so that the gene can be “conditionally” disrupted in mice. This is the first time a conditional Has2 knockout mouse has been created, a breakthrough that opens vast possibilities for future research. The paper was published online in the journal Development on July 24.

HA is a large sugar molecule that is produced by every cell in the body and has been thought to play a role in joint disease, heart disease and invasive cancers. Yu Yamaguchi, M.D., Ph.D., a professor in the Sanford Children’s Health Research Center at Burnham and Robert Kosher, Ph.D., a professor in the Center for Regenerative Medicine and Skeletal Development at U.C.H.C. and colleagues showed that transgenic mice, in which Has2 was inactivated in the limb bud mesoderm, had shortened limbs, abnormal growth plates and duplicated bones in the fingers and toes.

“Because hyaluronic acid is so prevalent in the body, it has been difficult to study,” said Dr. Yamaguchi. “Systemic Has2 knockout mice died mid-gestation and could not be used to study the role of hyaluronan in adults. By inactivating Has2 in specific tissues, we give ourselves the opportunity to study the many roles hyaluronan plays in biology. This mouse model will be useful to study the role of hyaluronan in arthritis and skin aging, as well as cancer.”

MicroRNAs and HIV

Tariq Rana, Ph.D., director of the Program for RNA Biology at Burnham, and colleagues have discovered that specific microRNAs (non-coding RNAs that interfere with gene expression) reduce HIV replication and infectivity in human T cells.

In particular, miR29 plays a key role in controlling the HIV life cycle. The study suggests that HIV may have co-opted this cellular defense mechanism to help the virus hide from the immune system and antiviral drugs. The research was published on June 26 in the journal Molecular Cell.

The team found that the microRNA miR29 suppresses translation of the HIV-1 genome by transporting the HIV mRNA to processing bodies (Pbodies), where they are stored or destroyed. This results in a reduction of viral replication and infectivity. The study also showed that inhibition of miR29 enhances viral replication and infectivity. The scientists further demonstrated that strains of HIV-1 with mutations in the region of the genome that interact with miR29 are not inhibited by miR29.

“We think the virus may use this mechanism to modulate its own lifecycle, and we may be able to use this to our advantage in developing new drugs for HIV,” says Dr. Rana. “Retroviral therapies greatly reduce viral load but cannot entirely eliminate it. This interaction between HIV and miR29 may contribute to that inability. Perhaps, by targeting miR29, we can force HIV into a more active state and improve our ability to eliminate it.”

Carbohydrate Acts as Tumor Suppressor

Minoru Fukuda, Ph.D., and colleagues have discovered that specialized complex sugar molecules (glycans) that anchor cells into place act as tumor suppressors in breast and prostate cancers.

These glycans play a critical role in cell adhesion in normal cells, and their decrease or loss leads to increased cell migration by invasive cancer cells and metastasis. An increase in expression of the enzyme that produces these glycans, Â3GnT1, results in a significant reduction in tumor activity. The research was published July 6 in the journal Proceedings of the National Academy of Sciences.

The specialized glycans are capable of binding to laminin and are attached to the á-dystroglycan cell surface protein. This binding facilitates adhesion between the epithelium and basement membrane and prevents cells from migrating. The team demonstrated that Â3GnT1 controls the synthesis of laminin-binding glycans in concert with the genes LARGE/LARGE2. Downregulating Â3GnT1 reduces the amount of the glycans, leading to greater movement by invasive cancer cells. However, when the researchers forced aggressive cancer cells to express Â3GnT1, the lamininbinding glycans were restored and tumor formation decreased.

These results indicate that certain carbohydrates on normal cells and enzymes that synthesize those glycans, such as Â3GnT1, function as tumor suppressors,” says Dr. Fukuda. “Up regulation of Â3GnT1 may become a novel way to treat cancer.”

Caspase 8 and Invasive Cancer

Cancer Center director Kristiina Vuori, M.D., Ph.D., and colleagues have found that the Caspase-8 protein, long known to play a major role in promoting programmed cell death (apoptosis), helps relay signals that can cause cancer cells to proliferate, migrate and invade surrounding tissues.

The study was published in the journal Cancer Research on June 15.

The team showed that Caspase-8 caused neuroblastoma cancer cells to proliferate and migrate. For the first time, Caspase-8 was shown to play a key role in relaying the growth signals from epidermal growth factor (EGF) that cause cell division and invasion. The researchers also identified an RXDLL amino acid motif that controls the signaling from the EGF receptor through the protein kinase Src to the master cell proliferation regulator protein MAPK. This same signaling pathway stimulates neuroblastoma cells to migrate and invade neighboring tissues—a critical process in cancer metastasis.

“Caspase-8 has a well defined role in promoting apoptosis, especially in response to activation of the so-called death receptors on the outside of cells,” says Darren Finlay, Ph.D., first author on the paper. “Although Caspase-8 is involved in apoptosis, it is rarely deleted or silenced in tumors, suggesting that it was giving cancer cells a leg up in some other way.”

What Makes Stem Cells Tick

Investigators at Burnham and The Scripps Research Institute (TSRI) have made the first comparative, largescale phosphoproteomic analysis of human embryonic stem cells (hESCs) and their differentiated derivatives.

The data may help stem cell researchers understand the mechanisms that determine whether stem cells divide or differentiate, what types of cells they become and how to control those complex mechanisms to facilitate development of new therapies. The study was published in the August 6 issue of the journal Cell Stem Cell.

“While the field of stem cell biology has become accustomed to looking at changes in genes, we have come to realize that proteins are the real work horses and ultimately determine cell behavior,” says Evan Snyder, M.D., Ph.D., professor and director of Burnham’s Stem Cell and Regenerative Biology program. “This study represents the first comprehensive study of genes being activated during differentiation and offers predictions on cell behavior.”

Protein phosphorylation, the biochemical process that modifies protein activities by adding a phosphate molecule, is central to cell signaling. Using sophisticated phosphoproteomic analyses, the team of Laurence Brill, Ph.D., senior scientist at Burnham’s Proteomics Facility, Dr. Synder and Sheng Ding, Ph.D., associate professor at TSRI, catalogued 2,546 phosphorylation sites on 1,602 phosphoproteins. Prior to this research, protein phosphorylation in hESCs was poorly understood. Identification of these phosphorylation sites provides insights into known and novel hESC signaling pathways and highlights signaling mechanisms that influence selfrenewal and differentiation.

“This research will be a big boost for stem cell scientists,” said Dr. Brill. “The protein phosphorylation sites identified in this study are freely available to the broader research community, and researchers can use these data to study the cells in greater depth and determine how phosphorylation events determine a cell’s fate.”

Unraveling How Cells Respond to Low Oxygen KnutzenGary Chiang, Ph.D., and colleagues have elucidated how the stability of the REDD1 protein is regulated.

The REDD1 protein is a critical inhibitor of the mTOR signaling pathway, which controls cell growth and proliferation. The study was published in the August 2009 issue of EMBO Reports.

As part of the cellular stress response, REDD1 is expressed in cells under low oxygen conditions (hypoxia). The Burnham scientists showed that the REDD1 protein rapidly undergoes degradation by the ubiquitin-proteasome system, which allowed for the recovery of mTOR signaling once oxygen levels were restored to normal.

“Cells initially shut down the most energy-costly processes, such as growth, when they’re under hypoxic stress,” says Dr. Chiang. “They do this by expressing REDD1, which inhibits the mTOR pathway. But when the cell needs the mTOR pathway active, REDD1 has to be eliminated first. Because the REDD1 protein turns over so rapidly, it allows the pathway to respond very dynamically to hypoxia and other environmental conditions.”

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