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Transforming growth factor beta mediated programmed cell death in tissue regeneration of the murine intestinal mucosa
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Transforming growth factor beta mediated programmed cell death in tissue regeneration of the murine intestinal mucosa (Published in Gastroenterology 122: 1364-1375, 2002)
Nicole Drinker* Center of Anatomy,Department of Neuroanatomy,Georg August UniversityG6ttingen, 37075 G6ttingen, Germany
Summary. Programmed cell death (PCD) is a key phenomenon in regulating cell-numbers. Apoptosis is essentially required for a balanced homeostasis between cell proliferation and cell death in multicellular organisms. Apoptosis is especially relevant in the gastrointestinal tract, as the mammalian intestinal mucosa undergoes continual epithelia cell turnover. Growth factors like transforming growth factor beta (TGF-[3) appear to play key regulatory functions in a diverse spectrum of biological processes including regulation of proliferative activity, and, as discovered most recently, apoptosis. Several studies from our group indicated the pro-apoptotic role of endogenous TGF-fl. We have shown that PCD of chick ciliary, dorsal root, and spinal motor neurons is largely prevented following application of a neutralizing antibody that recognizes all three TGF-13 isoforms [1]. The pro-apoptotic role of endogenous TGF-[~ was confirmed by showing TGF-[~'s capacity to prevent cell death in the developing nervous system, exemplified by the chick retina [2]. Besides, we reported on the role of TGF-~ in mediating apoptosis in interdigital webs of the developing mouse limb thereby indicating TGF-[3's requirement for PCD during morphogenesis of non-neuronal tissue [3]. To postulate a generalized mechanism of TGF-f3-mediated apoptosis, its role in the regulation of PCD during tissue regeneration needed to be determined. The gastrointestinal tract offers an ideal system to study apoptotic processes in vivo as cell proliferation in the crypts of Lieberktihn has to be counterbalanced by cell death at the villus tips. In the present study [4] we localized TGF-132 and TGF[~3 via Western Blot analysis and immunocytochemistry in
* Winner of the Wolfgang-Bargmann-Prize2003 Ann Anat (2003) 185:299-300 © Urban & FischerVerlag
http:llwww'urbanfischer'delj°urnalslannanat
the intestinal mucosa of three-week-old mice using isoform-specific anti-TGF-f3 antibodies. Western Blot analysis revealed a higher level of TGF-132 expression in the small intestine (compared to the colon) and an increased TGF-[~3 level in the colon (compared to the small intestine). Both isoforms exhibit prominent immunostaining along the villus axis and the crypts of Lieberktihn of the small intestine but were also detected in the colon. Immunoreactivity for TI~R-II was found to be somewhat more prominent at the villus tip, the region where apoptotic enterocytes are frequently found. Interestingly, we additionally observed an isoform-specific expression of endogenous TGF-[32 and TGF-[33 in the small intestine. As revealed by confocal laser scan microscopy and comparative PBA staining, endocrine cells stain intensely positive for TGF-~2, whereas TGF-f33 immunoreactivity was found predominantly in goblet cells. To address the question whether TGF-[~2 and TGF-~3 play a role in intestinal epithelium regeneration by regulation PCD of enterocytes we analyzed in guts of two heterozygous TGF-[3 mouse strains, deficient for either one TGF-~2 or TGF-~3 allel. Apoptotic levels quantified by TUNEL- and ELISA assays showed a significant reduction in cell death in the small intestine of Tgfff2 +/- and Tgfff3 +/- heterozygous mouse mutants. In the colon, however, apoptotic levels seem to be unaffected in mutant mice. This significant decrease in programmed cell death denotes a mentionable shift in proliferation/cell death balance which, however, was not counterbalanced by a reduction in cell proliferation but accompanied by an increase in villus length. To address the question how TGF-132 and TGF-133 mediate intestinal apoptosis mechanistically and if these isoforms also influence the levels of anti-apoptotic proteins in vivo, the expression of Bcl-2 and Bcl-XL in the 0940-9602/03/185/4-299 $15.00/0
murine intestine was assayed. Western blot analysis revealed that level of Bcl-XL and Bcl-2 is significantly upregulated in Tg)]32 +/- and TgJ]33 +/- mice. In the study presented [4] we established a key role of TGF-~2 and TGF~3 in mediating apoptosis if the intestinal mucosa. Our results gain special significance with respect to a generalized mechanism of TGF-~ mediated apoptosis in morphogenesis of neuronal and non-neuronal tissue as well as during tissue regeneration. Besides, although apoptosis is essential for the maintenance of normal gut epithelial function, a shift in proliferation/cell death balance might be a predisposition for tumerogenesis. The significance of apoptosis for intestinal disease currently focuses on carcinogenesis. It is conceivable that therapeutic strategies for treatment of intestinal cancer will be developed based on drugs interfering with cell proliferation and programmed cell death. On the other hand, application of neutralizing antibodies to TGF-~s may be effective in diseases that therapeutically require stimulation of epithelial regeneration and suppression of apoptosis.
References [1] Krieglstein K, Richter S, Farkas L, Schuster N, Dflnker N, Oppenheim RW, Unsicker K (2000) Reduction of endogenous transforming growth factors beta prevents ontogenetic neuron death. Nat Neurosci 3:1085-1090 [2] Drinker N, Schuster N, Krieglstein K (2001) TGF-~ modulates programmed cell death in the retina of the developing chick embryo. Development 128:1933-1942 [3] DUnker N, Sehmitt K, Krieglstein K (2002) TGF-~ is required for programmed cell death in interdigital webs of the developing mouse limb. Mech Dev 113:111-120 [4] Dt~nker N, Schmitt K, Schuster N, Krieglstein K (2002) The role of transforming growth factor beta 2 and 3 in mediating apoptosis in the murine intestinal mucosa. Gastroenterol 122:1364-1375
300
Transforming growth factor beta mediated programmed cell death in tissue regeneration of the murine intestinal mucosa (Published in Gastroenterology 122: 1364-1375, 2002)
Nicole Drinker* Center of Anatomy,Department of Neuroanatomy,Georg August UniversityG6ttingen, 37075 G6ttingen, Germany
Summary. Programmed cell death (PCD) is a key phenomenon in regulating cell-numbers. Apoptosis is essentially required for a balanced homeostasis between cell proliferation and cell death in multicellular organisms. Apoptosis is especially relevant in the gastrointestinal tract, as the mammalian intestinal mucosa undergoes continual epithelia cell turnover. Growth factors like transforming growth factor beta (TGF-[3) appear to play key regulatory functions in a diverse spectrum of biological processes including regulation of proliferative activity, and, as discovered most recently, apoptosis. Several studies from our group indicated the pro-apoptotic role of endogenous TGF-fl. We have shown that PCD of chick ciliary, dorsal root, and spinal motor neurons is largely prevented following application of a neutralizing antibody that recognizes all three TGF-13 isoforms [1]. The pro-apoptotic role of endogenous TGF-[~ was confirmed by showing TGF-[~'s capacity to prevent cell death in the developing nervous system, exemplified by the chick retina [2]. Besides, we reported on the role of TGF-~ in mediating apoptosis in interdigital webs of the developing mouse limb thereby indicating TGF-[3's requirement for PCD during morphogenesis of non-neuronal tissue [3]. To postulate a generalized mechanism of TGF-f3-mediated apoptosis, its role in the regulation of PCD during tissue regeneration needed to be determined. The gastrointestinal tract offers an ideal system to study apoptotic processes in vivo as cell proliferation in the crypts of Lieberktihn has to be counterbalanced by cell death at the villus tips. In the present study [4] we localized TGF-132 and TGF[~3 via Western Blot analysis and immunocytochemistry in
* Winner of the Wolfgang-Bargmann-Prize2003 Ann Anat (2003) 185:299-300 © Urban & FischerVerlag
http:llwww'urbanfischer'delj°urnalslannanat
the intestinal mucosa of three-week-old mice using isoform-specific anti-TGF-f3 antibodies. Western Blot analysis revealed a higher level of TGF-132 expression in the small intestine (compared to the colon) and an increased TGF-[~3 level in the colon (compared to the small intestine). Both isoforms exhibit prominent immunostaining along the villus axis and the crypts of Lieberktihn of the small intestine but were also detected in the colon. Immunoreactivity for TI~R-II was found to be somewhat more prominent at the villus tip, the region where apoptotic enterocytes are frequently found. Interestingly, we additionally observed an isoform-specific expression of endogenous TGF-[32 and TGF-[33 in the small intestine. As revealed by confocal laser scan microscopy and comparative PBA staining, endocrine cells stain intensely positive for TGF-~2, whereas TGF-f33 immunoreactivity was found predominantly in goblet cells. To address the question whether TGF-[~2 and TGF-~3 play a role in intestinal epithelium regeneration by regulation PCD of enterocytes we analyzed in guts of two heterozygous TGF-[3 mouse strains, deficient for either one TGF-~2 or TGF-~3 allel. Apoptotic levels quantified by TUNEL- and ELISA assays showed a significant reduction in cell death in the small intestine of Tgfff2 +/- and Tgfff3 +/- heterozygous mouse mutants. In the colon, however, apoptotic levels seem to be unaffected in mutant mice. This significant decrease in programmed cell death denotes a mentionable shift in proliferation/cell death balance which, however, was not counterbalanced by a reduction in cell proliferation but accompanied by an increase in villus length. To address the question how TGF-132 and TGF-133 mediate intestinal apoptosis mechanistically and if these isoforms also influence the levels of anti-apoptotic proteins in vivo, the expression of Bcl-2 and Bcl-XL in the 0940-9602/03/185/4-299 $15.00/0
murine intestine was assayed. Western blot analysis revealed that level of Bcl-XL and Bcl-2 is significantly upregulated in Tg)]32 +/- and TgJ]33 +/- mice. In the study presented [4] we established a key role of TGF-~2 and TGF~3 in mediating apoptosis if the intestinal mucosa. Our results gain special significance with respect to a generalized mechanism of TGF-~ mediated apoptosis in morphogenesis of neuronal and non-neuronal tissue as well as during tissue regeneration. Besides, although apoptosis is essential for the maintenance of normal gut epithelial function, a shift in proliferation/cell death balance might be a predisposition for tumerogenesis. The significance of apoptosis for intestinal disease currently focuses on carcinogenesis. It is conceivable that therapeutic strategies for treatment of intestinal cancer will be developed based on drugs interfering with cell proliferation and programmed cell death. On the other hand, application of neutralizing antibodies to TGF-~s may be effective in diseases that therapeutically require stimulation of epithelial regeneration and suppression of apoptosis.
References [1] Krieglstein K, Richter S, Farkas L, Schuster N, Dflnker N, Oppenheim RW, Unsicker K (2000) Reduction of endogenous transforming growth factors beta prevents ontogenetic neuron death. Nat Neurosci 3:1085-1090 [2] Drinker N, Schuster N, Krieglstein K (2001) TGF-~ modulates programmed cell death in the retina of the developing chick embryo. Development 128:1933-1942 [3] DUnker N, Sehmitt K, Krieglstein K (2002) TGF-~ is required for programmed cell death in interdigital webs of the developing mouse limb. Mech Dev 113:111-120 [4] Dt~nker N, Schmitt K, Schuster N, Krieglstein K (2002) The role of transforming growth factor beta 2 and 3 in mediating apoptosis in the murine intestinal mucosa. Gastroenterol 122:1364-1375
300