Contact Information: Harvard Institutes of Medicine, 77 Avenue Louis Pasteur, Room 570, Boston, MA 02115

Telephone: (617) 525-5820

Fax: (617) 525-5821

Email: mhediger@rics.bwh.harvard.edu

Description of Research:

The goal of our research is to elucidate the molecular and functional characteristics of nutrient transporters, their implications in human physiology and diseases, and their applications to drug therapy. Using expression cloning with Xenopus oocytes, we identified and characterized transporters of various classes. These include transporters of iron, vitamin C, urea, citrate, glutamate, dibasic amino acids and peptides, as well as epithelial calcium channels and a neutral solute channel. The current focus of our research is directed toward those transporters with highest medical implications. These include transporters of iron, calcium, vitamin C and glutamate. Studies involve structure-function analysis, biophysical characterization, regulation of transporters, and transgenic experiments. Using a multi disciplinary approach we are investigating the distribution of expression and physiological implications of transporters in major organs such as intestine, kidney and brain. We are also unraveling the involvement of transporters in human diseases such as cancer, kidney stone disease, hemochromatosis and amyotrophic lateral sclerosis.

Current Projects:

1. Molecular physiology of iron transport

Iron is vital for most processes of life. The cloning and characterization of the mammalian divalent metal ion transporter DMT1 in our laboratory in 1997 was the first demonstration of an active cellular uptake mechanism for Fe²⁺ (See Article). Expression studies of DMT1 revealed that this protein is a H+-coupled cotransporter of several divalent metal ions, including Fe²⁺, Zn²⁺, Mn²⁺, Co²⁺, Cd²⁺ and Ni²⁺. DMT1 is crucial for normal intestinal absorption of iron as well as iron utilization in developing red blood cells in which iron is required for hemoglobin synthesis (See Article). Iron is also essential to the normal function of many enzymes. Our data show that DMT1 is expressed in the intestinal brush border membrane, and that closely related splice variants are expressed in many other organs including kidney, liver and brain.

DMT1 plays a critical role in the pathogenesis of iron deficiency and overload disorders. A missense mutation, G185R, was identified in DMT1 as the cause of iron deficiency anemia in rodents. Hereditary hemochromatosis is one of the most common genetic diseases in which there is excessive intestinal iron absorption and a build up of iron to toxic levels in several organs. Our recent studies using intestinal biopsy samples revealed that upregulation of DMT1 is tightly associated with the pathogenesis of hereditary hemochromatosis (See Article). This upregulation results in excessive iron absorption and toxic deposition in major organs such as liver and heart. Dysregulation of DMT1 mRNA expression is likely the cause of abnormal body iron sensing due to mutations in the HFE hemochromatosis gene. My laboratory has shown that intestinal DMT1 mRNA levels are highly regulated and responsive to changes in body iron status, in part via post-transcriptional regulation involving an iron responsive element in the 3'-non-translated region of DMT1 mRNA (See Article). This regulatory mechanism appears to be disturbed in hemochromatosis.

In addition to DMT1-mediated uptake of inorganic iron in the intestine, there is a second pathway for heme-iron absorption. In fact, heme iron, found in meat, poultry and fish, is two to three times more absorbable than non-heme iron found in plant-based foods. The absorption of heme iron, however, is still poorly understood and my goal is to identify the proteins involved. The aims are to clarify the detailed molecular mechanism of this process, to study how it is regulated to supply appropriate amounts of this essential micronutrient, and to determine what the role of heme iron transport is in patients with iron-deficiency anemia and hemochromatosis.

We are currently using biochemical and electrophysiological approaches to reveal the transport mechanisms of DMT1. Of particular interest are the questions of how exactly DMT1 is coupled to protons, what the molecular basis is for transport-associated ion leaks and what the structural determinants are which define metal ion selectivity.

2. Epithelial calcium channels and calcium homeostasis

Calcium is the major inorganic component of the skeleton and an essential messenger in signal transduction in all living cells. The apical calcium entry channel CaT1 (TRPV6) was identified in our laboratory by expression cloning in 1999 (See Article) (See Article) (See Article). Together with its kidney homologue, CaT2 (TRPV5, also known as ECaC) (See Article), these channels form a distinct, highly calcium-selective subgroup of the TRP family of cation channels. CaT1 and CaT2 play a central role in total body calcium homeostasis and their regulation directly affects intestinal calcium absorption, renal calcium excretion and bone metabolism (See Article). We recently showed that the CaT1 and CaT2 channels are regulated by vitamin D and other steroid hormones. Thus, understanding the biology and mechanisms of regulation of these channels is of vital importance. We are also interested in developing therapeutic applications using CaT1 and CaT2 as targets, since these channels appear to be candidate molecules for drug development to alter intestinal and renal calcium transport, for example, in patients with kidney stone disease.

Our laboratory has elucidated the channel pore properties and tissue distribution of CaT1 and CaT2. Our recent studies also demonstrated that CaT1 and CaT2 mRNA expression is 1,25-vitamin D regulated, with >10-fold increases for CaT1 and 2-4 fold increases for CaT2 (See Article). The higher responsiveness of intestinal CaT1 is consistent with the much larger fluctuations of luminal calcium in the intestine compared to kidney.

To further understand the role of CaT1 in calcium absorption and homeostasis, we established knockout mice which are currently under investigation (manuscript in preparation). Thus far, our data reveal the following: CaT1 knockout males and females are less fertile than their +/- or +/+ littermates. A significant percentage of mice born to CaT1 KO mothers develop alopecia and eventually also dermatitis. In vivo oral gavage studies with 45Ca revealed that CaT1 KO mice have deficient intestinal Ca2+ absorption and a defect in renal reabsorption, resulting in urinary Ca2+ wasting. CaT1 KO mice have a significant decrease in bone mineral density, even in the presence of high amounts of Ca2+ in their diet, which is probably due to Ca2+ wasting by their kidneys.

Fig. 1. Ca2+ homeostasis. Ca2+ levels in blood and other extracellular fluids are tightly controlled mainly through the actions of the parathyroid hormone (PTH) (1)) (Fig. 1). A decrease in plasma Ca2+ levels is detected by the calcium sensing receptor (CASR) in parathyroid cells, resulting in release of PTH. Circulating PTH then stimulates renal reabsorption and bone resorption of Ca2+ and, in conjunction with renal conversion of vitamin D into its active form, 1,25-dihydroxyvitamin D, in up-regulation of intestinal Ca2+ absorption. 1,25-vitamin D is known to act on gene transcription through a nuclear steroid receptor, resulting in increased Ca2+ absorption in the proximal small intestine.

Biological variabilities in intestinal calcium absorption and renal reabsorption are crucial determinants of Ca²⁺ homeostasis. We hypothesize that polymorphisms in the CaT1 and CaT2 genes favor hypercalciuria and renal stone formation and we are planning to perform a rigorous genetic examination of the role of these genes in idiopathic hypercalciuria.

3. The vitamin C transporters SVCT1 and SVCT2 (SLC23A1 and SLC23A2)

It has been known for more than 70 years that vitamin C is essential to the human body and the disease scurvy, caused by an extreme deficiency of vitamin C, has been recognized for hundreds of years. Further, Nobel prizes winner Linus Pauling postulated that high doses of vitamin C are effective against colds and other illnesses. Despite these facts, until a few years ago, little was known about how the body actually absorbs vitamin C, and how the vitamin is distributed to the many organs at appropriate concentrations to ensure crucial enzymatic reactions (e.g. collagen synthesis) and to protect tissues and organs from oxidative damage. This processes requires the vitamin C transporters SVCT1 and SVCT2 recently identified in our laboratory (See Article).

Localization studies showed that SVCT1 is largely confined to absorbing epithelia such as intestine and kidney where it serves whole body homeostasis. In contrast, SVCT2 accounts for the widespread tissue-specific uptake of vitamin C. Plasma vitamin C concentrations are maintained between 10-160µM and any excessive intake of the vitamin will be excreted by the kidney. In contrast, its concentration is at least 100 times higher in tissues which require the vitamin for biochemical reactions or protection against oxidative damage, such as adrenal glands, pituitary gland, thymus, corpus luteum, retina and cornea. Vitamin C therefore needs to be distributed in a regulated manner into organs at the appropriate concentration, and this requires specific, concentrative transport processes such as SVCT2. The cloning of these transporters enables us to examine 1) how they are regulated to ensure an appropriate supply of the vitamin to various tissues, 2) how these transporters are involved in protection against oxidative stress, and 3) whether there are ways to specifically upregulate SVCT2 as a strategy to protect against viral infections, such as the common cold. To determine whether the lack of SVCT2 results in scurvy-like symptoms, Nussbaum and colleagues established SVCT2 knockout mice (See Article). Interestingly, these mice died within a few minutes of birth with respiratory failure and extensive intracerebral hemorrhage. These studies highlight the central importance of vitamin C in protecting the body against oxidative damage, especially during fetal and neonatal development.

4. Role of glutamate transporters in amyotrophic lateral sclerosis (ALS)

ALS is a highly lethal disease, causing selective degeneration of motor neurons and paralysis. About 10% of cases are inherited as an autosomal dominant trait, and ~20% of these are caused by mutations in the cytosolic Cu²⁺/Zn²⁺ superoxide dismutase SOD1. My hypothesis is that damage of glutamate transporters by oxygen free radicals leads to neurotoxic accumulation of extracellular glutamate following its release at glutamatergic synapses and death of motor neurons. We tested this hypothesis using an electrophysiological approach to monitor glutamate transporter activity as well as transfected cells which express both glutamate transporters and ALS-linked mutant SOD1. The experiments demonstrated that the target for oxidative damage is the glutamate transporter protein GLT1 (See Article ). Our findings reinforce the logic behind the use of antioxidants and riluzole (a blocker of voltage-activated Na⁺ channels that affects glutamate release) in slowing the course of sporadic ALS. Further research is in progress to uncover the source of oxidative stress in patients with sporadic ALS and to obtain the basic knowledge necessary to design improved therapeutic strategies.

SLC series of Human Transporter Gene Families:

Currently, there are 44 human transporter gene families belonging to the SLC (solute carrier) series. These families include more than 300 human transporter genes. Over the past decade, I have served as a specialty advisor to the HUGO Gene Nomenclature Committee and was in charge of all assignments. I also established a WEB site with detailed information about these genes: http://www.bioparadigms.org.

Human Membrane Transporter Mini-Review Series:

I served as the guest editor of the European Journal of Physiology special issue (vol. 447, no. 5, February 2004) with articles on each SLC transporter family. The articles include summaries on potential therapeutic implications of membrane transporters.

International BioMedical Transporter Conferences:

To promote the pharmaceutical discovery in the transporter field, I have, together with Peter Meier-Abt from the University of Zürich, established a series of biannual international conferences in Switzerland – see WEB site: http://www.bioparadigms.org. The next conference will be held in August 2005 in the Olma Congress Center in St. Gallen, Switzerland. It will be entitled “BioMedical Transporters 2005: Bridging basic and applied sciences”.