Lorne Protein: Jack Martin's Cheshire cat

The Leach lecturer at the 2005 Lorne Protein conference has a persistent streak, Graeme O'Neill discovers.

Jack Martin, emeritus professor of medicine at the University of Melbourne, has devoted the greater part of an illustrious research career to the dogged pursuit of a personal Cheshire cat called PTHrP.

Several times in the mid-1970s, the St Vincent's Medical Research Institute biochemist caught it smiling enigmatically from his antibody tests on serum and tissue samples from cancer patients with hypercalcaemia.

Martin had set out to determine why some patients with lung and kidney cancers, breast cancer and a several other forms of cancer, develop dangerously elevated levels of calcium in their blood. He began his search in the early days of the recombinant DNA revolution. It would become a classic scientific detective story that has evolved with the science, and new technology.

Martin has been honoured with an invitation to deliver the Leach Lecture at the 2005 Lorne Conference on Protein Structure and function at Philip Island's Continental Hotel on February 6.

Citation laureate

Last year the International Citation Index named Martin its Australian citation laureate in biochemistry. His research papers, which have illuminated the mechanisms of bone remodelling, osteoporosis and cancer-induced hypercalcaemia, have been cited more than 7000 times by other research teams during the past 20 years.

Martin's initial suspicion was that the cancers that cause hypercalcaemia were over-expressing parathyroid hormone (PTH), a hormone normally secreted by parathyroid gland. PTH was known for its ability to mobilise calcium from its main reservoir in bone, but his research over the next decade would rule out PTH as the instigator of cancer-associated hypercalcaemia.

By the early 1980s, he had strong evidence that an unidentified hormone-like agent was mobilising bone calcium and causing life-threatening hypercalcaemia in some cancer patients.

His research group, then based at the Repatriation Hospital in Heidelberg, developed an extremely sensitive cellular assay for PTH, based on cultured cells from a rat model of osteosarcoma. The cells respond to PTH by producing cyclic AMP in a dose-dependent manner.

Martin grew cells from a lung cancer patient who had died from the effects of high blood calcium, and treated samples of the culture medium with PTH-neutralising antibodies. The neutralised media still induced vigorous production of cyclic AMP in their rat osteosarcoma cells.

Mystery hormone

The mystery hormone was obviously targeting the same receptor as PTH. It accelerates bone resorption, ramping up calcium levels in the bloodstream, but also reprograms the kidneys to prevent the excess calcium being excreted. Patients with cancer-associated hypercalcaemia lose bone mass, and develop the brittle-bone disorder osteoporosis.

Martin's antibody tests in the 1970s hinted that his Cheshire cat of a hormone not only mimicked PTH, but that the two shared a degree of peptide homology. For these reasons, it was dubbed parathyroid hormone-resembling hormone (PTHrH).

Over the next five years Dr Jane Mosely (now at University of Melbourne at St Vincent's Hospital) helped Martin to purify PTHrP from the lung-cancer cell culture medium. Dick Wettenhall (now director of the Bio21 Institute in Melbourne) began sequencing the molecule, and by 1987 had a lead: the first 20 residues from the amino 'head' of the molecule. Eight of the first 13 residues matched up with the PTH peptide sequence -- explaining the 'fuzzy positives' from Martin's earlier PTH antibody tests. But beyond that point, the 'tail' of PTHrP bore no resemblance to that of PTH.

Wettenhall's 20 residues provided enough DNA-sequence information to mount a search for the PTHrH gene, but when it bogged down, Martin and Wettenhall sought help from Genentech in the US.

Evolutionary relationship

"Genentech cloned PTHrP only a few days after Dick finished sequencing the first 50 amino acids," Martin said. "They showed a structural similarity and biological activity similar to PTH, so it seemed likely there was an evolutionary relationship."

Soon after, Martin and his colleagues learned that PTHrP was on chromosome 12, while PTH was on chromosome 11. A long list of neighbouring genes, including IGF-1 and IGF-2 (insulin-like growth factor), conform to the same pattern. Such synteny points to an ancient duplication event, probably involving a segment of an ancestral chromosome 11.

The PTHrP gene features nine protein-coding exons, with alternative splicings giving rise to at least three different forms of the protein. PTH has just three exons, which code for a single protein.

In evolutionary terms, PTH might be more accurately characterised as PTHrP-like gene, chipped from an old 'block' of chromosome 11. Tests on the Japanese puffer fish Fugu ribripes offer more evidence that PTHrP was the archetypal gene -- antibodies detect PTHrP, but no PTH.

The cat materialises

After more than a decade of research, Martin had succeeded in materialising his Cheshire cat. "It seemed we could now explain the malignancy thing [hypercalcaemia]," he said. "We did some experiments in an animal model, and assayed the serum of some cancer patients, and confirmed that PTHrP was elevated in patients suffering from cancer-induced hypercalcaemia."

The PTHrP tale became more intriguing when Martin's team ran antibody tests on tissue samples from throughout the body. Despite PTHrP's hormone-like activity when expressed by tumours, they found is not a classical hormone. A variety of human and animal tissues secrete PTHrP: bone marrow, the vascular endothelium, the gastric epithelium, smooth muscle, the uterine endothelium and skin keratinocytes.

In these tissues, PTHrP is locally synthesised, and exerts purely local effects. Among other things, it relaxes the vascular endothelium and smooth muscle, two effects long attributed to PTH. In bone marrow, locally secreted PTHrP stimulates the differentiation and activation of osteoclasts, the specialised cells that break down and recycle bone.

But PTHrP is virtually undetectable in the bloodstream, leading Martin's team to conclude they were dealing with a paracrine factor, not a hormone. Cancer excepted, there is only one other time when PTHrP becomes detectable in the bloodstream: during late pregnancy and lactation. "It's produced in large amounts by the lactating breast," Martin said. "The concentration in milk is a couple of hundred nanograms per millilitre - as it turns out, we could probably have purified enough from 2 litres of milk to sequence the first 100 amino acids."

Original role

Martin believes this was the protein's original role: to mobilise calcium from the mother's bones and transfer it across the placenta, to build the bones of the developing foetus -- and subsequently to maintain a steady flow of calcium to the newborn, via the mammary gland.

Dr Andrew Karaplis of Canada's McGill University developed a PTHrP knockout mouse -- the mutation is perinatally lethal in newborn mice. They die from severe compression of the rib cage, which restricts heart and lung development. Mice heterozygous for the knockout mutation survive, but within a few months show inadequate bone formation, resulting in osteoporosis.

A striking finding from the knockout study is that female mice fail to develop nipples and mammary glands -- PTHrP may have been a key player the divergence of mammals from reptilian ancestors in the Permian period, more than 250 million years ago.

The mouse models have provided insights into a rare human genetic syndrome, Blomstrand's chondrodysplasia, caused by loss-of-function mutations -- not in the human PTHrP gene, but in the gene for its receptor, the G-coupled protein receptor PTHR1. The mutation is perinatally lethal; babies that survive are born severely deformed, and survive only a few months. The syndrome is typified by cardiovascular defects, shortened limbs, and severe cranio-facial abnormalities, including malformed mandibles.

Foetuses with Blomstrand's chondrodysplasia also lack nipples and mammary glands, and have severely impacted teeth, due to the failure of osteoclasts to clear a path through the bone of the jaw, for the teeth to erupt.

'Exquisitely regulated'

Martin said PTHrP must be "exquisitely regulated" to exert its local effects. The best evidence for this is its paradoxical action on bone remodelling: while it stimulates osteoclasts to break down bone and mobilise calcium, at a specific dose and frequency, it does the reverse: it stimulates bone formation. Its mimic, PTH, is actually being marketed in this role, as an injectable treatment for osteoporosis.

A single, daily injection resulting in a short, sharp peak in the bloodstream stimulates bone formation, yet chronic administration induces bone resorption. Exactly the same thing happens with PTHrP. Martin said new findings from Canadian researchers suggest that the commercial PTH therapy mimics the local, paracrine effects of PTHrP on bone remodelling.

The new osteoporosis therapy was almost 75 years in the making. Martin said researchers recognised in the 1930s that PTH caused bone resorption, but could actually promote bone formation when given as a single, acute dose. But the discovery faded from memory as sedentary habits and the rise of the motor vehicle led to the modern epidemic of osteoporosis.

There is life still in the Martin's venerable cat. The peptide homology in the first 34 residues of PTH and PTHrP allows both molecules to interact with the same receptor, but sequences towards the middle of the protein regulate placental transport of calcium. And the protein's carboxy terminal also exhibits novel biological activity unrelated to the protein's central role in calcium metabolism.

For good measure, says Martin, the protein also contains a nuclear-localisation sequence, suggesting that, in addition to its hormone and paracrine functions, it has a direct role in gene regulation. While the target genes have yet to be identified, PTHrP is produced during cell division -- not only is it critical to in the life cycle of mammals, it may prove to have a vital role in the cell cycle too.