3S-HMGB1, THE PROTEIN THAT ACCELERATES TISSUE REGENERATION

HMGB1 is a protein with a thousand faces. Normally present in the nucleus of cells (where it binds to DNA, it promotes the repair and transcription of genes), under conditions of cellular stress it can be released into the cytoplasm or out of the cell, where it performs many different functions, perhaps not yet all identified.

When a tissue is damaged, the processes of inflammation – mediated by the immune system – and subsequent regeneration of the tissue are essential for healing. In a research published in The Journal of Experimental Medicine, a group of researchers identified HMGB1 as the key-protein that orchestrates both processes. Not only that: by modifying HMGB1 in the laboratory they have created a version that only favours regeneration. The new protein, called 3S-HMGB1, accelerates the process of tissue regeneration in mice that suffered muscle or liver damage. The study was coordinated by the Doctors Emilie Vénéreau and Mario Tirone, researchers at the “Chromatin Dynamics” Unit at the San Raffaele Hospital, and by Prof. Marco Bianchi, Professor of Molecular Biology at our University and Head of the Unit itself.

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“The HMGB1 protein (High Mobility Group 1) has been known and studied for a long time, but most research has always focused on its role in inflammation and how to block the protein to inhibit the inflammatory process” explains Dr. Vénéreau. “HMGB1 is released whenever a stress or damage occurs in a tissue: this release works as an alarm signal that recruits the cells of the immune system in the damaged site. Our idea was that it could also be important for tissue repair, the post-inflammation phase in which the stem cells reconstruct the tissue”.

Already in 2012 the group coordinated by Prof. Bianchi had shown that the different extracellular activities of HMGB1 depended on its oxidation state: when the protein is in its oxidized form, it promotes inflammation; vice versa its reduced (non-oxidized) form works as a booster signal for cells involved in tissue repair.

Why should the same protein induce inflammation on the one hand and regeneration on the other? Prof. Bianchi answers: “At the moment we do not know it with certainty, but we think there may be a utility: when you are injured and you suffer from a trauma, almost certainly an infection also occurs, which you need to eradicate before going on to regenerate the tissue. According to our model, the same protein decides whether there is an infection – and so it is not time to reconstruct the tissue – or if the infection is absent and you can proceed with tissue repair. It is still a hypothesis, but it seems reasonable to us”.

A SMALL CHANGE FOR A GREAT RESULT

“In this study we have created a modified form of the protein, called 3S-HMGB1: the new protein actually contains 3 amino acids (the “building blocks” that make up the protein) which have been changed to  serines (S), instead of the cysteines (C) normally present. “This new form is no longer oxidizable by the cell, and in this way it allows the tissues to be repaired) without however inducing inflammation. We have shown this in the muscle and liver of mice”.

Simplified diagram of the three forms of HMGB1 and their functions. On the left, the oxidized form of HMGB1 has a disulfide bridge that joins two sulfur atoms (S), and causes inflammation, recruiting all of the immune system cells and tissue damage. In the center, the reduced form of HMGB1 (with three -SH groups associated with cysteines (C) in the protein body) promotes tissue regeneration. On the right, the form modified by the research group of Prof. Bianchi: 3S-HMGB1 has three serines (S) instead of cysteines (C) and cannot be oxidized, thus allowing tissue repair without the negative consequence of inflammation and tissue damage.
Simplified diagram of the three forms of HMGB1 and their functions. On the left, the oxidized form of HMGB1 has a disulfide bridge that joins two sulfur atoms (S), and causes inflammation, recruiting all of the immune system cells and tissue damage. In the center, the reduced form of HMGB1 (with three -SH groups associated with cysteines (C) in the protein body) promotes tissue regeneration. On the right, the form modified by the research group of Prof. Bianchi: 3S-HMGB1 has three serines (S) instead of cysteines (C) and cannot be oxidized, thus allowing tissue repair without the negative consequence of inflammation and tissue damage.

Dr. Vénéreau continues: “Other molecules described in the literature proved to be effective in tissue regeneration, but had to be administered several times a week (or even several times a day). The very encouraging data that emerged from our study are that a single 3S-HMGB1 injection is sufficient to accelerate two-fold regeneration of muscle and liver by acting respectively on the resident muscle stem cells or on the hepatocytes (the liver cells that perform the regenerative function in this organ)”.

Notes Dr. Tirone, first author of the study and PhD student of Silvia Brunelli, professor at the University of Milan-Bicocca and co-author of the work: “3S-HMGB1 is more stable than natural HMGB1, so much so that mice injected with this modified form give very similar and homogeneous responses. Perhaps this is also why it works better and that there is no need for further injections”.

Laboratory members of Prof. Bianchi’s lab (sixth from left, white shirt). Dr. Vénéreau is at the center of the photo (red shirt); Dr. Tirone is fourth from left (blue polo shirt).
Laboratory members of Prof. Bianchi’s lab (sixth from left, white shirt). Dr. Vénéreau is at the center of the photo (red shirt); Dr. Tirone is fourth from left (blue polo shirt).

AN EXPERIMENTATION (FOR NOW) ONLY ON ANIMALS

The results have been so far achieved only experimenting on mice; a clinical application on humans will take some time”, explains Dr. Vénéreau. What are the difficulties? “First we should find the right pathology in which to test the effectiveness of 3S-HMGB1, which is not a trivial matter. Initially our choice had fallen on bone diseases. The problem, however, is that there are no standards of cure for bone: bone fractures are so common and heterogeneous that it is difficult to objectively evaluate the actual validity of our molecule. Another difficulty that should not be underestimated is the need of funds for research”.

The application of 3S-HMGB1 on humans – adds Dr. Tirone – would give undoubted advantages: just think of the post-operative hospitalization, which can be reduced thanks to faster tissue regeneration. This would lead shorter hospitalization and a reduction in costs for hospitals and the National Health System”.

Representative image of tibialis anterior after 7 days from damage. HMGB1 (Red) is located in the nucleus, Laminin (green) contours the muscle fibers. Nuclei in blue. Courtesy of Prof. Bianchi's group.
Representative image of tibialis anterior after 7 days from damage. HMGB1 (Red) is located in the nucleus, Laminin (green) contours the muscle fibers. Nuclei in blue. Courtesy of Prof. Bianchi's group.

To date, the only therapeutic approaches under study are aimed at completely neutralizing the activity of the HMGB1 protein, in particular following a trauma or in inflammatory pathologies, eliminating in this way both its pro-inflammatory and regenerative effects. “Our research suggests instead that selectively inhibiting its oxidation would be a decidedly more promising therapeutic approach. This would allow to limit the presence of the oxidized form, deleterious, favoring on the contrary the reduced, regenerative form. Although we are far from using the 3S-HMGB1 in patients, focusing on the inhibition of HMGB1 oxidation could have more immediate applications”.

The understanding of HMGB1’s dual nature could have important implications also in the field of oncology, where on the contrary it is the inflammatory action of the natural protein to have a potential therapeutic role. “Tumors behave similarly to damaged tissuesadds Prof. Bianchi. “In this case the regenerative action of HMGB1 can however be harmful. Exploiting its inflammatory properties, we could instead hinder the growth of the tumor”.

Prof. Bianchi and Dr. Vénéreau took part in an Italian documentary about epigenetics, winner of an important award in the USA. Read more here!

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