March 2010

Jerry Lingrel: Pumping Out Great Science

 

Sci-Focus---Lingrel
Jerry Lingrel’s science revolves around blood. Photo by Dan Davenport, University of Cincinnati AHC

For Jerry B. Lingrel, a distinguished professor in the department of molecular genetics, biochemistry and microbiology at the University of Cincinnati and an associate editor for the Journal of Biological Chemistry, great science is in the blood.

Not that Lingrel comes from a strong scientific pedigree— to the contrary, he was just a small-town Midwestern boy whose desire to pursue a career in science was encouraged by his parents— but rather that Lingrel has made a living studying this vital fluid. From his early breakthrough in isolating and characterizing globin messenger RNA to his subsequent work sequencing the sodium-potassium pump (Na,K-ATPase) and studying its role in regulating blood pressure to his discovery of Krüppel-like factor 2, a transcription factor that, among other things, maintains blood vessel integrity; Lingrel’s science revolves around blood.

And, it all began not by design but simply by convenience.

In 1968, shortly after Lingrel, an Ohio native, had returned home to begin a professorship at the University of Cincinnati after a postdoctoral fellowship at the California Institute of Technology, he decided to focus on the messenger RNA that encoded the proteins involved in protein synthesis.

As he recalls, mRNAs were sort of a “black box” at that time, about a decade after their initial discovery. “They had been identified in bacteria,” Lingrel says, “and everyone believed they were present in eukaryotic cells as well. But, it was still a lot of theory because no one had managed to isolate an individual mRNA that coded for a specific protein.”

Lingrel wasn’t sure if it would be feasible to isolate mRNAs, but he figured, “If I want to give myself a chance, I might as well find a system where mRNA is abundant.”

That’s where blood came in to the picture. During his postdoc at Caltech with Henry Borsook, a pioneer in protein synthesis studies, Lingrel had worked extensively with reticulocytes (immature red blood cells), which were an ideal model system: As hemoglobin factories, all they basically do is churn out globin chains all day long.

By that same token, Lingrel figured reticulocytes should contain a vast amount of α and β globin mRNAs. To isolate these mRNAs, he thought that using an Escherichia coli cell-free translating system, similar to that employed by Marshall Nirenberg to crack the genetic code, would be a reasonable approach; Lingrel would add different fractions of total mouse reticulocyte RNA to extracts of E. coli translation machinery and see which ones produced globins.

Of course, his initial attempts didn’t bear fruit, and he jokes, “It was not the most ideal way to discover that there are significant differences between bacterial and eukaryotic protein synthesis.”

However, when he changed his approach and used a cell-free translation system from rabbit reticulocytes, he achieved success and identified a 9 S RNA that resulted in the synthesis of both α and β globin. In doing this, he identified and translated the first mammalian messenger RNA.

Sci-Focus---mRNA-hybridization

Jerry Lingrel recently has discovered a role for Na,K-ATPase in the brain, as highlighted by this in situ mRNA hybridization analysis of the ion transporter’s α1 and α2 isoforms in developing mouse brains; A and B show sections through the hippocampus, with the star highlighting robust expression for α1 but not α2 in the choroid plexus, whereas the arrow highlights the more intense α2 presence in the ependymal lining. C and D show sections through the cortex, with the arrow highlighting the strong expression of α2 in the pia mater. Moseley, A. E. et al, J. Biol. Chem. (2003) 278, 5317–5324.

No Tension Here

Over the next few years, Lingrel and his lab would further characterize globin mRNA, uncovering many of the key features we know about these transcripts, including their 5’ CAP structure and 3’ poly-A tail as well as the fact that mature mRNAs in the cytoplasm arose from larger precursor RNA molecules in the nucleus. Through his approach, which he shared with many colleagues, globin synthesis became the model in which to study mRNA structure and translation.

As the 1970s progressed, Lingrel continued his work with globins. Having made complementary DNA to his isolated mRNA, he was able to identify and clone globin genes from a variety of mammalian cells and study both the changes in globin expression during development and the evolution of the globin gene family.

Then, in 1976, the University of Cincinnati’s microbiology department hired a new faculty member, Dennis Lang, who ended up becoming one of Lingrel’s neighbors. Given their similar destination, the two ended up carpooling on numerous occasions. And through this ridesharing, Lingrel was introduced to another molecular black box: the Na,K-ATPase, which pumps potassium into and sodium out of a cell.

“Lang had done his graduate studies under Efraim Racker at Cornell, who was a leading expert on the Na,K-ATPase,” Lingrel says, “and, during our rides, he kept telling me how important this enzyme was in biology. So, one day I asked him what they knew about the ATPase structure, and he responded that they didn’t even know the amino acid sequence yet.”

That sparked an idea: Lingrel admittedly knew very little about transport proteins, but what he did know was molecular cloning.

Sci-Focus---Sperm-A
Sci-Focus---Sperm-B

One of Jerry Lingrel’s many discoveries regarding Na,K-ATPase was uncovering the sperm-specific α4 isoform, revealed in A as being localized to the mid-piece region of the flagellum. B shows a control sperm with only secondary antibody staining. Woo, A. L., et al. J. Biol. Chem. (2000) 275, 20693–20699.

“I thought to myself: Oh, our lab could handle that; and if this ATPase is as important as Lang believes, then sequencing and characterizing it would be a tremendous advance to science.”

Working with membrane proteins would be tricky, but Lingrel always has operated with a simple belief: If you take the time to think about a problem and pick the right system to work on it, there’s no reason a project should not work. So, he picked sheep kidney cells, an abundant source of Na,K-ATPase, and, together with a very talented postdoc, Gary Shull, he managed to isolate ATPase mRNA, make cDNA clones, decipher the amino acid sequence for the α and β subunits and identify important residues for pump activity. 

“It ended up that we published our ATPase sequence in the exact same issue of Nature that David MacLennan reported his sequence for the calcium ATPase,” Lingrel says. “So, in one day, we managed to open up a whole new era in the study of ion transport proteins.”

Lingrel would go on to make countless more discoveries regarding Na,K-ATPase, including showing that the catalytic α subunit had four separate isoforms which contributed to the numerous functions the pump had throughout the body. For example, the α4 isoform is found exclusively in sperm and helps sperm cells move, whereas the α2 and α3 isoforms are highly expressed in brain cells and produce learning deficiencies when knocked out in mice. 

Some of his most valued work, however, involves the central role of the ATPase in sodium transport in hypertension.

It long had been known that steroid-like compounds like ouabain and digitoxin (both derived from plants) could block the Na,K-ATPase and, thus, force increased heart contractions, a fact that was used to develop similar compounds as treatments for congestive heart failure (though they are no longer widely used, as better drugs such as angiotensin-converting enzyme (ACE) inhibitors have come along).

Out of Focus: A Little Less Conversation, A Little More Science

Jerry Lingrel’s sabbatical with John Gurdon at the MRC laboratory was a fantastic experience, although it did take a while to adjust to local customs. “The scientists there loved to socialize,” he says. “They talked over morning coffee, lunch and afternoon tea.” At first, Lingrel didn’t join the fun – “I thought I was here to work not talk”— but he eventually got hooked. “I had so much fun talking to all the great scientific minds at the MRC about ideas and techniques, and soon I understood why experiments never seemed to fail there.” However, Lingrel ended up becoming such the social butterfly during the day that he had to come back to lab every night to do the actual experiments.

However, some controversy also developed, as evidence seemed to suggest that animals produced their own ouabain-like steroids to modulate Na,K-ATPase activity (so-called endogenous ouabains). As Lingrel notes, while several studies had shown the presence of ouabain in normal blood samples, it was extremely difficult to prove that they had been synthesized in the body. “And, from a logical standpoint, people wondered why the human body would want to synthesize ouabain, which is a toxic molecule,” Lingrel says.

On the other hand, comparative work done by Lingrel’s team found that the binding site for ouabain and similar drugs in the Na,K-ATPase α subunit was heavily conserved from fruit flies to humans, which would support a physiological role, and, by extrapolation, a physiological ligand.

“So, we decided to work on this mystery, but, rather than focus on the compounds, we decided to focus on the binding site.” His reasoning was based on the interesting observation that one of the four mouse α subunit isoforms (α1) happened to be resistant to ouabain. So, he mutated two amino acids in the α2 isoform (the major vascular form), so it resembled α1 in one group of mice and altered α1 so it resembled α2 in another group. He then induced stress in the mice to see what happened.

“Sure enough, the mice that contained the mutated α2 were resistant to hypertension,” Lingrel says, “whereas the animals that had the altered α1 almost blew up because they became so hypertensive.” That seemed to confirm that the ouabain binding site was physiologically important in regulating blood pressure, and something in the blood was interacting with it. Lingrel is currently collaborating with some colleagues at the University of Cincinnati to try to find that elusive endogenous ligand.

Low-stress Levels

In some ways, Lingrel is still the curious, small-town Midwestern boy who was fascinated with science and nature. Some of it may arise from the fact that he never really left Ohio. Except for his two-year postdoc at Caltech and a one-year sabbatical at the MRC laboratory in Cambridge, England, Lingrel has been a steady fixture in the Buckeye state, from growing up in Byhalia, to his college years at Otterbein College in Westerville, to his graduate studies at The Ohio State University and finally his long and impressive professorship at the University of Cincinnati.

It’s that continued connection with his youth, spending countless hours trying to understand how things work, that has shaped his research path, be it his globin or Na,K-ATPase studies, or even his more recent foray into KLF2 (Krüppel-like factor).

These studies began as an offshoot of his research with globin expression, but, as in the case of the Na,K-ATPase, Lingrel notes, “I’ve managed to make second careers out of what I thought would just be side projects.”

Other researchers had discovered a transcription factor, called the erythroid Krüppel-like factor, that was critical in orchestrating the switch from fetal to adult hemoglobin expression, and Lingrel used some cDNA from EKLF as a clone to screen for other proteins that bound to hemoglobin genes.

What he uncovered was not just a protein that shared a similar gene-binding region, but one that closely resembled EKLF, ushering in a new Krüppel-like protein family.

Sci-Focus---sagittal-sections

Jerry Lingrel’s group recently has shown that KLF2 is involved in making blood vessels. Here, stained sagittal sections of wild type (A) and knock-out (B) mouse aorta highlight the vascular defects that occur when KLF2 is deficient. Wu, J., Bohanan, C. S., Neumann, J. C., and Lingrel, J. B., J. Biol. Chem. (2008) 283, 3942–3950.

He named this new transcription factor LKLF (for lung Krüppel-like factor, although it was later renamed KLF2) and began pursuing its role. He found that it was vital for proper development of the lung and other tissues in embryos but also had a role in the formation, maturation and integrity of blood vessels.

The most interesting aspect of KLF2 function, though, was that it was induced in endothelial cells by fluid shear stress. “That caught my eye,” Lingrel says, “because it’s in areas of low-shear stress, like bifurcations, where you get plaque buildups and atherosclerosis; so KLF2 might be an atheroprotective agent.” His group is currently developing transgenic mice that overexpress KLF2 in low-stress areas and testing their resistance to plaque buildup when fed a high-fat diet.

Lingrel is thrilled that his work has helped so many other scientists. Although he’s focused primarily on the vascular system, both KLF2 and the Na,K-ATPase are fairly ubiquitous, and his fundamental discoveries are applicable to areas like muscle activity, neuroscience and development. And, not surprisingly, he’s been flooded with requests for advice or one of his many transgenic mouse lines, which he’s always happy to oblige.

One cannot help but wonder whether it’s perfectly fitting or somewhat ironic that this calm and contented man is revealing the mechanisms of atherosclerosis and hypertension. 

References

Lockard, R. E., and Lingrel, J. B. (1971) Identification of Mouse Haemoglobin Messenger RNA. Nature, 233, 204–206.

Shull, G. E., Schwartz, A., and Lingrel, J. B. (1985) Amino-acid Sequence of the Catalytic Subunit of the (Na+/K+) ATPase Deduced from a Complementary DNA. Nature, 316, 691–695.

Anderson, K. P., Kern, C. B., Crable, S. C., and Lingrel, J. B. (1995) Isolation of a Gene Encoding a Functional Zinc Finger Protein Homologous to Erythroid Krüppel-like Factor: Identification of a New Multigene Family. Mol. Cell. Biol. 15, 5957–5965.

Woo, A. L., James, P. F., and Lingrel, J. B. (2000) Sperm Motility Is Dependent on a Unique Isoform of the Na,K-ATPase. J. Biol. Chem. 275, 20693–20699.

Dostanic-Larson, I., Van Huysse, J. W., Lorenz, J. N., and Lingrel, J. B. (2005) The Highly Conserved Cardiac Glycoside Binding Site of Na,K-ATPase Plays a Role in Blood Pressure Regulation. Proc. Natl. Acad. Sci. U.S.A. 102, 15845–15850.

Huddleson, J. P., Ahmad, N., and Lingrel, J. B. (2006) Up-regulation of the KLF2 Transcription Factor by Fluid Shear Stress Requires Nucleolin. J. Biol. Chem. 281, 15121–15128.

Nick Zagorski (nzagorski@asbmb.org) is a science writer at ASBMB.

 


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