- Email: [email protected]
Regeneration: Every Clot Has a Thrombin Lining
Current Biology, Vol. 13, R517–R518, July 1, 2003, ©2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S0960-9822(03)00444-5
Regeneration: Every Clot Has a Thrombin Lining Malcolm Maden
Why do some amphibian organs regenerate and others do not? And why do most mammalian organs not regenerate? New work on lens regeneration indicates that the answer lies in the need for cells to dedifferentiate first, and that what makes them dedifferentiate may be the presence of thrombin in combination with cellular clotting factors.
Some vertebrates, such as the Urodeles or tailed amphibians, have an incredible ability to regenerate a range of organs and tissues, even as adults. They can regenerate limbs, tails, spinal cords, jaws, gills, parts of the brain, retinas, irises, lenses and sections of the heart [1,2]. But even within this group of animals there are variations in this ability: for example, the newt can regenerate its lens, but the axolotl cannot, and the Anurans (frogs and toads) lose the ability to regenerate limbs as they approach metamorphosis. The existence of these varying abilities presents us with a golden opportunity to compare regenerating and non-regenerating organs to search for vital cellular and molecular differences which may explain regenerative behaviour. The ultimate intent, of course, is to induce regeneration in a non-regenerating organ, such as the mammalian limb, spinal cord or heart. A way of stimulating the intrinsic regenerative abilities of a mammalian organ would solve all those difficult ethical issues involved in using foetal cells or the immunological problems of using someone else’s stem cells. A major boost towards this optimistic goal has been provided in work published recently in Current Biology by Imokawa and Brockes [3], who have studied lens regeneration in newts. Following its removal from the eye, the newt lens regenerates and the new lens cells arise exclusively from the dorsal margin of the iris, not the ventral iris [4]. These dorsal pigmented epithelial cells dedifferentiate, lose their pigmentation, re-enter the cell cycle, divide and then redifferentiated as lens cells. Why do the dorsal iris cells do this but not the ventral? And why can newts do this while the closely related axolotl cannot? Amazingly, the answer seems to involve a chain of events all starting with clotting factors in the blood, in particular thrombin, and ending up in the nucleus with the phosphorylation of the retinoblastoma (Rb) protein. This work developed from studies of how newt muscle cells behave compared to their mammalian counterparts. After limb amputation, newt myotubes dedifferentiate by fragmenting into mononucleate cells, re-enter the cell cycle and participate in the formation MRC Centre for Developmental Neurobiology, New Hunt’s House, King’s College London, Guy’s Campus, London Bridge, London SE1 1UL, UK.
Dispatch
of the blastema, a conical shaped mound of cells which will replace all the missing parts of the limb. Clearly, some factor in the blastemal environment tells the myotubes — and the other cell types, such as cartilage and connective tissue — to do this, and the factor may be present in serum because re-entry to the cell cycle can be recapitulated in culture by simply adding serum [5]. The only mammalian muscle cells that behave like this are those from the retinoblastoma knockout mouse [6]. The Rb protein normally binds to the E2F family of transcription factors to prevent the transcription of genes which are required for entry into S phase of the cell cycle. In the absence of Rb, cellcycle progression would thus not be inhibited. In normal cells that can divide, the Rb protein becomes phosphorylated and dissociates from E2F, thus allowing cell-cycle progression, and this occurs in newt myotubes in culture. The nuclear event permitting myotube dedifferentiation thus involves Rb phosphorylation. But what is the signal in serum which starts it all off? It had previously been found that none of the known growth factors would stimulate cell cycle re-entry, but after fractionating serum, a thrombin fraction contained two activities, one of which was thrombin and the other a proteolytic product of thrombin [7]. Thrombin is not a direct-acting factor on the cells, because compounds that activate the thrombin receptor are not active in stimulating cell cycle re-entry of newt myotubes; so thrombin first needs to be activated by proteolysis to make the second product found in fractionated serum. Another coagulation factor, plasmin, also induced cell-cycle reentry, so it seems that the initial event involves proteolysis of an as yet unidentified serum component — called thrombin-derived activity (TDA) in Figure 1 — by either of these two coagulation factors. Does this happen in vivo, however? The answer seems to be yes because thrombin can be detected in the blastema after limb amputation in newts, and it is not just present immediately after amputation, when the blood is clotting, but is still high one week after amputation, when dedifferentiation and cell-cycle re-entry are occurring. And the most surprising result is reported in the recent work of Imokawa and Brockes [3]: they have found that thrombin activation can be detected at the dorsal margin of the iris, from where the new lens will regenerate, but not at the ventral margin which cannot regenerate the lens. When inhibitors of thrombin activity were injected into the eye, the dorsal iris cells did not enter S phase and lens regeneration was inhibited, providing crucial evidence for the vital role of thrombin. Furthermore, the closely related Urodele, the axolotl, which can regenerate limbs but not lenses, did not activate thrombin in the eye after iris removal, but did activate thrombin in the limb. So does this mean all we have to do is to throw thrombin onto mammalian limbs and they will regenerate? Unfortunately no, because all vertebrate
Dispatch R518
Amputate the limb or remove the lens
Serum prothrombin released from vasculature
Membrane tissue factor Activates
Dorsal iris cell or muscle cell
Thrombin Activates Induces cell cycle re-entry
TDAa
TDA from serum Current Biology
sera contain thrombin and there must be something special about the Urodele myotubes or dorsal iris cells in addition to the presence of thrombin. Imokawa and Brockes [3] suggest this could be a cellular component like Tissue Factor, an integral membrane protein which nucleates the formation of clotting factors that together activate prothrombin (Figure 1). Although there is still much to be discovered, this recent work holds out great promise for uncovering the reason why some tissues and organs can regenerate and others cannot. It is very intriguing that blood-borne clotting factors are assuming a new prominence, because early on in the long history of limb regeneration several investigators suggested that the blood was the source of the blastemal cells. Lymphocytes and/or macrophages were observed transforming into ‘polyblasts’ and fibroblasts [8,9]. It now seems, however, that it is the non-cellular part of the blood which is of crucial importance for regeneration. References 1. Goss, R.J. (1969). Principles of Regeneration. (New York: Acad. Press.) 2. Stocum, D.L. (1995). Wound Repair, Regeneration, and Artificial Tissues. (Austin : Springer-Verlag.) 3. Imokawa, Y., and Brockes, J.P. (2003). Selective activation of thrombin is a critical determinant for vertebrate lens regeneration. Curr. Biol. 13 May issue. 4. Eguchi, G. (1988). Cellular and molecular background of Wolffian lens regeneration. Cell Differ. Dev. 25(suppl), 147-158. 5. Tanaka, E.M., Gann, A.A.F., Gates, P.B., and Brockes, J.P. (1997). Newt myotubes reenter the cell cycle by phosphorylation of the retinoblastoma protein. J. Cell Biol. 136, 155-165. 6. Schneider, J.W., Gu, W., Zhu, L., Mahdavi, V., and Nadal-Ginard, B. (1994). Reversal of terminal differentiation mediated by p107 in Rb/- muscle cells. Science 264, 1467-1471. 7. Tanaka, E.M., Drechsel, D.N., and Brockes, J.P. (1999). Thrombin regulates S-phase re-entry by cultured newt myotubes. Curr. Biol. 9, 792-799. 8. Fritsch, C. (1911). Experimentelle studien uber Regenerationsvorganger des Gleidmassenskeletts. Zool. Jahrb. Abt. Physiol. 30, 377472. 9. Hellmich, W. (1931). Histology of regeneration in different species of adult and larval urodeles. Anat. Rec. 48, 303-307.
Figure 1. A scheme for the early events proposed to stimulate the dedifferentiation and cell cycle re-entry of limb or iris cells, and which are thus responsible for inducing regeneration. Limb amputation or lens removal results in the release of serum prothrombin, which is activated to thrombin by tissue factor present on the cell surface of dorsal iris cells or blood cells at the limb amputation plane. Thrombin activates an unknown activity in serum — thrombinderived activity (TDA) — producing TDAa, which feeds back on the dorsal iris or amputated limb cells to induce cell-cycle re-entry and consequent dedifferentiation. Thus both the thrombin and a cell-based component are required for the induction of regeneration.
Regeneration: Every Clot Has a Thrombin Lining Malcolm Maden
Why do some amphibian organs regenerate and others do not? And why do most mammalian organs not regenerate? New work on lens regeneration indicates that the answer lies in the need for cells to dedifferentiate first, and that what makes them dedifferentiate may be the presence of thrombin in combination with cellular clotting factors.
Some vertebrates, such as the Urodeles or tailed amphibians, have an incredible ability to regenerate a range of organs and tissues, even as adults. They can regenerate limbs, tails, spinal cords, jaws, gills, parts of the brain, retinas, irises, lenses and sections of the heart [1,2]. But even within this group of animals there are variations in this ability: for example, the newt can regenerate its lens, but the axolotl cannot, and the Anurans (frogs and toads) lose the ability to regenerate limbs as they approach metamorphosis. The existence of these varying abilities presents us with a golden opportunity to compare regenerating and non-regenerating organs to search for vital cellular and molecular differences which may explain regenerative behaviour. The ultimate intent, of course, is to induce regeneration in a non-regenerating organ, such as the mammalian limb, spinal cord or heart. A way of stimulating the intrinsic regenerative abilities of a mammalian organ would solve all those difficult ethical issues involved in using foetal cells or the immunological problems of using someone else’s stem cells. A major boost towards this optimistic goal has been provided in work published recently in Current Biology by Imokawa and Brockes [3], who have studied lens regeneration in newts. Following its removal from the eye, the newt lens regenerates and the new lens cells arise exclusively from the dorsal margin of the iris, not the ventral iris [4]. These dorsal pigmented epithelial cells dedifferentiate, lose their pigmentation, re-enter the cell cycle, divide and then redifferentiated as lens cells. Why do the dorsal iris cells do this but not the ventral? And why can newts do this while the closely related axolotl cannot? Amazingly, the answer seems to involve a chain of events all starting with clotting factors in the blood, in particular thrombin, and ending up in the nucleus with the phosphorylation of the retinoblastoma (Rb) protein. This work developed from studies of how newt muscle cells behave compared to their mammalian counterparts. After limb amputation, newt myotubes dedifferentiate by fragmenting into mononucleate cells, re-enter the cell cycle and participate in the formation MRC Centre for Developmental Neurobiology, New Hunt’s House, King’s College London, Guy’s Campus, London Bridge, London SE1 1UL, UK.
Dispatch
of the blastema, a conical shaped mound of cells which will replace all the missing parts of the limb. Clearly, some factor in the blastemal environment tells the myotubes — and the other cell types, such as cartilage and connective tissue — to do this, and the factor may be present in serum because re-entry to the cell cycle can be recapitulated in culture by simply adding serum [5]. The only mammalian muscle cells that behave like this are those from the retinoblastoma knockout mouse [6]. The Rb protein normally binds to the E2F family of transcription factors to prevent the transcription of genes which are required for entry into S phase of the cell cycle. In the absence of Rb, cellcycle progression would thus not be inhibited. In normal cells that can divide, the Rb protein becomes phosphorylated and dissociates from E2F, thus allowing cell-cycle progression, and this occurs in newt myotubes in culture. The nuclear event permitting myotube dedifferentiation thus involves Rb phosphorylation. But what is the signal in serum which starts it all off? It had previously been found that none of the known growth factors would stimulate cell cycle re-entry, but after fractionating serum, a thrombin fraction contained two activities, one of which was thrombin and the other a proteolytic product of thrombin [7]. Thrombin is not a direct-acting factor on the cells, because compounds that activate the thrombin receptor are not active in stimulating cell cycle re-entry of newt myotubes; so thrombin first needs to be activated by proteolysis to make the second product found in fractionated serum. Another coagulation factor, plasmin, also induced cell-cycle reentry, so it seems that the initial event involves proteolysis of an as yet unidentified serum component — called thrombin-derived activity (TDA) in Figure 1 — by either of these two coagulation factors. Does this happen in vivo, however? The answer seems to be yes because thrombin can be detected in the blastema after limb amputation in newts, and it is not just present immediately after amputation, when the blood is clotting, but is still high one week after amputation, when dedifferentiation and cell-cycle re-entry are occurring. And the most surprising result is reported in the recent work of Imokawa and Brockes [3]: they have found that thrombin activation can be detected at the dorsal margin of the iris, from where the new lens will regenerate, but not at the ventral margin which cannot regenerate the lens. When inhibitors of thrombin activity were injected into the eye, the dorsal iris cells did not enter S phase and lens regeneration was inhibited, providing crucial evidence for the vital role of thrombin. Furthermore, the closely related Urodele, the axolotl, which can regenerate limbs but not lenses, did not activate thrombin in the eye after iris removal, but did activate thrombin in the limb. So does this mean all we have to do is to throw thrombin onto mammalian limbs and they will regenerate? Unfortunately no, because all vertebrate
Dispatch R518
Amputate the limb or remove the lens
Serum prothrombin released from vasculature
Membrane tissue factor Activates
Dorsal iris cell or muscle cell
Thrombin Activates Induces cell cycle re-entry
TDAa
TDA from serum Current Biology
sera contain thrombin and there must be something special about the Urodele myotubes or dorsal iris cells in addition to the presence of thrombin. Imokawa and Brockes [3] suggest this could be a cellular component like Tissue Factor, an integral membrane protein which nucleates the formation of clotting factors that together activate prothrombin (Figure 1). Although there is still much to be discovered, this recent work holds out great promise for uncovering the reason why some tissues and organs can regenerate and others cannot. It is very intriguing that blood-borne clotting factors are assuming a new prominence, because early on in the long history of limb regeneration several investigators suggested that the blood was the source of the blastemal cells. Lymphocytes and/or macrophages were observed transforming into ‘polyblasts’ and fibroblasts [8,9]. It now seems, however, that it is the non-cellular part of the blood which is of crucial importance for regeneration. References 1. Goss, R.J. (1969). Principles of Regeneration. (New York: Acad. Press.) 2. Stocum, D.L. (1995). Wound Repair, Regeneration, and Artificial Tissues. (Austin : Springer-Verlag.) 3. Imokawa, Y., and Brockes, J.P. (2003). Selective activation of thrombin is a critical determinant for vertebrate lens regeneration. Curr. Biol. 13 May issue. 4. Eguchi, G. (1988). Cellular and molecular background of Wolffian lens regeneration. Cell Differ. Dev. 25(suppl), 147-158. 5. Tanaka, E.M., Gann, A.A.F., Gates, P.B., and Brockes, J.P. (1997). Newt myotubes reenter the cell cycle by phosphorylation of the retinoblastoma protein. J. Cell Biol. 136, 155-165. 6. Schneider, J.W., Gu, W., Zhu, L., Mahdavi, V., and Nadal-Ginard, B. (1994). Reversal of terminal differentiation mediated by p107 in Rb/- muscle cells. Science 264, 1467-1471. 7. Tanaka, E.M., Drechsel, D.N., and Brockes, J.P. (1999). Thrombin regulates S-phase re-entry by cultured newt myotubes. Curr. Biol. 9, 792-799. 8. Fritsch, C. (1911). Experimentelle studien uber Regenerationsvorganger des Gleidmassenskeletts. Zool. Jahrb. Abt. Physiol. 30, 377472. 9. Hellmich, W. (1931). Histology of regeneration in different species of adult and larval urodeles. Anat. Rec. 48, 303-307.
Figure 1. A scheme for the early events proposed to stimulate the dedifferentiation and cell cycle re-entry of limb or iris cells, and which are thus responsible for inducing regeneration. Limb amputation or lens removal results in the release of serum prothrombin, which is activated to thrombin by tissue factor present on the cell surface of dorsal iris cells or blood cells at the limb amputation plane. Thrombin activates an unknown activity in serum — thrombinderived activity (TDA) — producing TDAa, which feeds back on the dorsal iris or amputated limb cells to induce cell-cycle re-entry and consequent dedifferentiation. Thus both the thrombin and a cell-based component are required for the induction of regeneration.