Rino Donato - Academia.edu (original) (raw)

Papers by Rino Donato

Gastroenterology

Background: The sequential proliferation, lineage-specific differentiation, crypt-villus migratio... more Background: The sequential proliferation, lineage-specific differentiation, crypt-villus migration, and cell death of the epithelial cells of the intestinal mucosa is tightly regulated by regulatory peptides, differentiation signals, and luminal stimuli, including nutrients and pathogenic/commensal organisms. Despite its importance for understanding normal homeostasis and pathogenesis of disease states, the intracellular signal transduction mechanisms involved remain incompletely understood. Here, we tested the hypothesis that PKD1 plays a key role in intestinal crypt cell proliferation In Vivo. Results: To clarify the role of PKD1 in intestinal epithelial cell proliferation In Vivo, we generated transgenic mice that express elevated PKD1 protein in the distal small intestinal and proximal colonic epithelium. The catalytic activity and multi-site phosphorylation (Ser744, Ser748 and Ser916) of PKD1 was strikingly higher in extracts from ileal mucosa of transgenic mice as compared with non-transgenic littermates. These results indicate that transgenic PKD1 is functional in the intestinal epithelium. PKD1 signaling stimulated intestinal cell proliferation, as shown by detection of 5-bromo-2-deoxyuridine (BrdU) incorporated into the cell nuclei of crypt cells of the ileum and proximal colon, where PKD1 protein is maximally expressed. Our results demonstrated a highly statistically significant increase (p<0.005) in DNA synthesizing cells in the crypts of the PKD1 transgenic mice as compared with non-transgenic littermates. In the intestine, normal cell numbers are maintained by balancing rates of cell proliferation, differentiation, migration and apoptosis. Consequently, we determined whether transgenic PKD1 leads to a change in tissue architecture, manifested by an increase in the size and total number of epithelial cells in the crypts. We measured crypt height (in micrometer and cell number) and crypt circumference (in micrometer and cell number) in histological sections of control and PKD1 transgenic mice. The data was used to calculate the size of individual cells and the total number of cells per crypt. Our results show a significant increase in the depth (either in μm or in number of cells) and in the total number of cells per crypt in the transgenic PKD1 mice as compared with the nontransgenic littermates (276 total cells per ileal crypt in transgenic mice versus 192 in nontransgenic mice; p< 0.005). These results indicate that the expression of the PKD1 transgene led to a marked increase (44%) in the total number of intestinal epithelial cells per crypt. Conclusion: Transgenic PKD1 expression increases the number of cells per crypt by stimulating the rate of crypt cell proliferation. These results support the hypothesis that PKD1 signaling plays a role in a pathway leading to proliferation in intestinal epithelial cells.

Digestive Diseases and Sciences, 2012

Growth of the small intestine in the infant rat is promoted by crypt fission and later by increas... more Growth of the small intestine in the infant rat is promoted by crypt fission and later by increased crypt cell proliferation. Notch signaling could promote crypt fission. Hes-1 is a Notch target gene. We assessed the effect of Notch signaling on intestinal crypt fission and on growth of the intestine in the infant rat. Hes-1 expression was determined in the small intestine of litters of Hooded Wistar rats aged between 3 and 72 days. Hes-1 RNA expression was measured by quantitative RT-PCR. Four groups of rats (n = 8 or 9) were injected daily, ip, either with vehicle or with the Notch inhibitor DAPT at doses of 3, 10, and 30 mg/kg, from days 9 to 13 of life, and killed on day 14. A microdissection technique was used to measure crypt fission, mitotic count, and apoptotic count. Data were analyzed by ANOVA and by use of Dunnett&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s F test. Hes-1 expression and crypt fission peaked on day 14. DAPT reduced Hes-1 immunostaining in proportion to dose. DAPT reduced villous area to 72 % (p &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.01), 53 % (p &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.001), and 38 % (p &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.001) of control values for 3, 10 and 30 mg/kg doses, respectively, and reduced crypt fission to 53 % (p &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.001) and 38 % (p &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.001) of control values, respectively, for 10 and 30 mg/kg doses. Crypt mitotic count was not affected by any DAPT dose. DAPT at 10 and 30 mg/kg significantly increased apoptosis in crypts, by 6.5 and 4.8-fold, respectively. We conclude that Notch signaling promotes crypt fission and growth of the intestine by maintaining low apoptosis of crypt cells.

International Conference on the Comparative Biology of the Monocotyledons (2nd: 1998: Sydney, Australia) Monocots II, 2000

Journal of Pediatric Gastroenterology and Nutrition, 2012

Objectives: Intestinal crypt fission peaks during infancy. In human and experimental familial pol... more Objectives: Intestinal crypt fission peaks during infancy. In human and experimental familial polyposis coli, increased crypt fission is due to activation of Wnt/b-catenin signalling, but the molecular basis of crypt fission during intestinal growth has not been examined. The aim of this project was to investigate whether crypt fission and intestinal growth are affected by experimental blockade of the Wnt/b-catenin signalling pathway. Methods: Hooded Wistar rats were given either the Wnt inhibitor, dickkopf (30 and 100 ng), daily or vehicle control intraperitoneally from days 11 to 15 and were killed at day 16. Intestinal morphometry was used to measure villous area, crypt area, percentage of crypt fission, and crypt mitotic count. Intestinal stem cells were assessed by expression of real time-polymerase chain reaction for Lgr5 (a stem cell marker), and the number of b-cateninexpressing crypts by immunostaining was determined after 100-ng dickkopf treatment. Results: Dickkopf at 30 and 100 ng/day reduced villous area to 71% (P ¼ 0.013) and 29% (P < 0.0001), crypt area to 42% (P ¼ 0.0026) and 30% (P ¼ 0.0067), and crypt fission to 51% (P ¼ 0.006) and 29% (P < 0.0001), respectively, of control values. Mitotic count per crypt did not change. Lgr5 RNA expression and the number of b-catenin-expressing crypts decreased in dickkopf-treated animals. Conclusions: We conclude that intestinal crypt fission during infancy is mediated by Wnt signalling. It is possible that local treatment with Wnt agonists could be used to increase intestinal growth.

The integrity, or barrier function, of the intestinal epithelium is of paramount importance in -m... more The integrity, or barrier function, of the intestinal epithelium is of paramount importance in -maintaining good health. This is largely imparted by a single layer of epithelial cells linked by the transmembrane tight junction protein complex near their apical surface. Disruption of epithelial permeability via the tight junctions can contribute to disease progression. The cytokine IFNγ is involved in many inflammatory processes and has been shown to dramatically increase permeability via changes at the tight junction in experimental models. One of its key effectors is the transcription factor, -IRF-1. In our studies of the role of IRF-1 in barrier function using the human T84 intestinal epithelial cell monolayer model, we have found that induction of IRF-1 alone is insufficient to change permeability and that if IRF-1 is involved in mediating the permeability effects of IFNγ, then other factors must also be required.

Biochimica Et Biophysica Acta-molecular Cell Research, 2009

The epithelial tight junction forms a barrier to paracellular solute movement. In this study we s... more The epithelial tight junction forms a barrier to paracellular solute movement. In this study we show that the heterotrimeric G-protein Gα13 regulates the epithelial tight junction barrier. We generated MDCKII kidney epithelial cell lines in which the expression of an active Gα13 mutant (Gα13Q226L) could be induced. We demonstrated that Gα13Q226L expression increased paracellular permeability and caused the disruption and redistribution of proteins comprising the tight junction and the adherens junction away from sites of cell contact and the appearance of basal stress fibers. The effects on the junctional proteins and the actin cytoskeleton were abrogated by the Rho kinase inhibitor Y27632 but not by the Src kinase inhibitor PP2. The Gα13 mediated increase in permeability was also Src kinase independent but was partly dependent on Rho kinase signalling. Our data establish a link between Gα13, Rho kinase signaling and epithelial barrier function and not only demonstrate that Gα13 regulates epithelial apical junction properties but that it does so via signaling pathways that are distinct from the closely related protein Gα12. j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b b a m c r

Gastroenterology

Background: The sequential proliferation, lineage-specific differentiation, crypt-villus migratio... more Background: The sequential proliferation, lineage-specific differentiation, crypt-villus migration, and cell death of the epithelial cells of the intestinal mucosa is tightly regulated by regulatory peptides, differentiation signals, and luminal stimuli, including nutrients and pathogenic/commensal organisms. Despite its importance for understanding normal homeostasis and pathogenesis of disease states, the intracellular signal transduction mechanisms involved remain incompletely understood. Here, we tested the hypothesis that PKD1 plays a key role in intestinal crypt cell proliferation In Vivo. Results: To clarify the role of PKD1 in intestinal epithelial cell proliferation In Vivo, we generated transgenic mice that express elevated PKD1 protein in the distal small intestinal and proximal colonic epithelium. The catalytic activity and multi-site phosphorylation (Ser744, Ser748 and Ser916) of PKD1 was strikingly higher in extracts from ileal mucosa of transgenic mice as compared with non-transgenic littermates. These results indicate that transgenic PKD1 is functional in the intestinal epithelium. PKD1 signaling stimulated intestinal cell proliferation, as shown by detection of 5-bromo-2-deoxyuridine (BrdU) incorporated into the cell nuclei of crypt cells of the ileum and proximal colon, where PKD1 protein is maximally expressed. Our results demonstrated a highly statistically significant increase (p<0.005) in DNA synthesizing cells in the crypts of the PKD1 transgenic mice as compared with non-transgenic littermates. In the intestine, normal cell numbers are maintained by balancing rates of cell proliferation, differentiation, migration and apoptosis. Consequently, we determined whether transgenic PKD1 leads to a change in tissue architecture, manifested by an increase in the size and total number of epithelial cells in the crypts. We measured crypt height (in micrometer and cell number) and crypt circumference (in micrometer and cell number) in histological sections of control and PKD1 transgenic mice. The data was used to calculate the size of individual cells and the total number of cells per crypt. Our results show a significant increase in the depth (either in μm or in number of cells) and in the total number of cells per crypt in the transgenic PKD1 mice as compared with the nontransgenic littermates (276 total cells per ileal crypt in transgenic mice versus 192 in nontransgenic mice; p< 0.005). These results indicate that the expression of the PKD1 transgene led to a marked increase (44%) in the total number of intestinal epithelial cells per crypt. Conclusion: Transgenic PKD1 expression increases the number of cells per crypt by stimulating the rate of crypt cell proliferation. These results support the hypothesis that PKD1 signaling plays a role in a pathway leading to proliferation in intestinal epithelial cells.

Digestive Diseases and Sciences, 2012

Growth of the small intestine in the infant rat is promoted by crypt fission and later by increas... more Growth of the small intestine in the infant rat is promoted by crypt fission and later by increased crypt cell proliferation. Notch signaling could promote crypt fission. Hes-1 is a Notch target gene. We assessed the effect of Notch signaling on intestinal crypt fission and on growth of the intestine in the infant rat. Hes-1 expression was determined in the small intestine of litters of Hooded Wistar rats aged between 3 and 72 days. Hes-1 RNA expression was measured by quantitative RT-PCR. Four groups of rats (n = 8 or 9) were injected daily, ip, either with vehicle or with the Notch inhibitor DAPT at doses of 3, 10, and 30 mg/kg, from days 9 to 13 of life, and killed on day 14. A microdissection technique was used to measure crypt fission, mitotic count, and apoptotic count. Data were analyzed by ANOVA and by use of Dunnett&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s F test. Hes-1 expression and crypt fission peaked on day 14. DAPT reduced Hes-1 immunostaining in proportion to dose. DAPT reduced villous area to 72 % (p &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.01), 53 % (p &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.001), and 38 % (p &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.001) of control values for 3, 10 and 30 mg/kg doses, respectively, and reduced crypt fission to 53 % (p &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.001) and 38 % (p &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.001) of control values, respectively, for 10 and 30 mg/kg doses. Crypt mitotic count was not affected by any DAPT dose. DAPT at 10 and 30 mg/kg significantly increased apoptosis in crypts, by 6.5 and 4.8-fold, respectively. We conclude that Notch signaling promotes crypt fission and growth of the intestine by maintaining low apoptosis of crypt cells.

International Conference on the Comparative Biology of the Monocotyledons (2nd: 1998: Sydney, Australia) Monocots II, 2000

Journal of Pediatric Gastroenterology and Nutrition, 2012

Objectives: Intestinal crypt fission peaks during infancy. In human and experimental familial pol... more Objectives: Intestinal crypt fission peaks during infancy. In human and experimental familial polyposis coli, increased crypt fission is due to activation of Wnt/b-catenin signalling, but the molecular basis of crypt fission during intestinal growth has not been examined. The aim of this project was to investigate whether crypt fission and intestinal growth are affected by experimental blockade of the Wnt/b-catenin signalling pathway. Methods: Hooded Wistar rats were given either the Wnt inhibitor, dickkopf (30 and 100 ng), daily or vehicle control intraperitoneally from days 11 to 15 and were killed at day 16. Intestinal morphometry was used to measure villous area, crypt area, percentage of crypt fission, and crypt mitotic count. Intestinal stem cells were assessed by expression of real time-polymerase chain reaction for Lgr5 (a stem cell marker), and the number of b-cateninexpressing crypts by immunostaining was determined after 100-ng dickkopf treatment. Results: Dickkopf at 30 and 100 ng/day reduced villous area to 71% (P ¼ 0.013) and 29% (P < 0.0001), crypt area to 42% (P ¼ 0.0026) and 30% (P ¼ 0.0067), and crypt fission to 51% (P ¼ 0.006) and 29% (P < 0.0001), respectively, of control values. Mitotic count per crypt did not change. Lgr5 RNA expression and the number of b-catenin-expressing crypts decreased in dickkopf-treated animals. Conclusions: We conclude that intestinal crypt fission during infancy is mediated by Wnt signalling. It is possible that local treatment with Wnt agonists could be used to increase intestinal growth.

The integrity, or barrier function, of the intestinal epithelium is of paramount importance in -m... more The integrity, or barrier function, of the intestinal epithelium is of paramount importance in -maintaining good health. This is largely imparted by a single layer of epithelial cells linked by the transmembrane tight junction protein complex near their apical surface. Disruption of epithelial permeability via the tight junctions can contribute to disease progression. The cytokine IFNγ is involved in many inflammatory processes and has been shown to dramatically increase permeability via changes at the tight junction in experimental models. One of its key effectors is the transcription factor, -IRF-1. In our studies of the role of IRF-1 in barrier function using the human T84 intestinal epithelial cell monolayer model, we have found that induction of IRF-1 alone is insufficient to change permeability and that if IRF-1 is involved in mediating the permeability effects of IFNγ, then other factors must also be required.

Biochimica Et Biophysica Acta-molecular Cell Research, 2009

The epithelial tight junction forms a barrier to paracellular solute movement. In this study we s... more The epithelial tight junction forms a barrier to paracellular solute movement. In this study we show that the heterotrimeric G-protein Gα13 regulates the epithelial tight junction barrier. We generated MDCKII kidney epithelial cell lines in which the expression of an active Gα13 mutant (Gα13Q226L) could be induced. We demonstrated that Gα13Q226L expression increased paracellular permeability and caused the disruption and redistribution of proteins comprising the tight junction and the adherens junction away from sites of cell contact and the appearance of basal stress fibers. The effects on the junctional proteins and the actin cytoskeleton were abrogated by the Rho kinase inhibitor Y27632 but not by the Src kinase inhibitor PP2. The Gα13 mediated increase in permeability was also Src kinase independent but was partly dependent on Rho kinase signalling. Our data establish a link between Gα13, Rho kinase signaling and epithelial barrier function and not only demonstrate that Gα13 regulates epithelial apical junction properties but that it does so via signaling pathways that are distinct from the closely related protein Gα12. j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b b a m c r