anil bhushan - Academia.edu (original) (raw)

Papers by anil bhushan

OBJECTIVEType 1 Diabetes (T1D) results from progressive loss of pancreatic beta cells due to auto... more OBJECTIVEType 1 Diabetes (T1D) results from progressive loss of pancreatic beta cells due to autoimmune destruction. We recently reported that during the natural history of T1D in humans and the female nonobese diabetic (NOD) mouse model, beta cells acquire a senescence-associated secretory phenotype (SASP) that is a major driver of disease onset and progression, but the mechanisms that activate SASP in beta cells were not explored. The objective of this study was to identify transcriptional mechanisms of SASP activation in beta cells.METHODSWe used the female NOD mouse model of spontaneous autoimmune T1D and ex vivo experiments on NOD mouse and human islets to test the hypothesis that Bromodomain Extra-Terminal domain (BET) proteins activate the beta cell SASP transcriptional program.RESULTSHere we show that beta cell SASP is transcriptionally controlled by BET proteins, including BRD4. Chromatin analysis of key beta cell SASP genes in NOD islets revealed binding of BRD4 at active ...

Journal of Clinical Investigation, 2019

Nature Cell Biology, 2019

In the version of this article originally published, the Gene Expression Omnibus (GEO) accession ... more In the version of this article originally published, the Gene Expression Omnibus (GEO) accession number listed in the data availability section was incorrectly given as GSE10979 instead of GSE109795. The sentence should read "RNA-seq data that support the findings of this study have been deposited in the Gene Expression Omnibus (GEO) under accession code GSE109795, " and the code should link to https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE109795. The error has been corrected in the HTML and PDF versions of the paper.

Nature Cell Biology, 2019

Despite advances in the differentiation of insulin-producing cells from human embryonic stem cell... more Despite advances in the differentiation of insulin-producing cells from human embryonic stem cells, the generation of mature functional β cells in vitro has remained elusive. To accomplish this goal, we have developed cell culture conditions to closely mimic events occurring during pancreatic islet organogenesis and β cell maturation. In particular, we have focused on recapitulating endocrine cell clustering by isolating and reaggregating immature β-like cells to form islet-sized enriched β-clusters (eBCs). eBCs display physiological properties analogous to primary human β cells, including robust dynamic insulin secretion, increased calcium signalling in response to secretagogues, and improved mitochondrial energization. Notably, endocrine cell *

Diabetes, 2018

The sustained expression of the MAFB transcription factor in human islet β-cells represents a dis... more The sustained expression of the MAFB transcription factor in human islet β-cells represents a distinct difference in mice. Moreover, mRNA expression of closely related and islet β-cell–enriched MAFA does not peak in humans until after 9 years of age. We show that the MAFA protein also is weakly produced within the juvenile human islet β-cell population and that MafB expression is postnatally restricted in mouse β-cells by de novo DNA methylation. To gain insight into how MAFB affects human β-cells, we developed a mouse model to ectopically express MafB in adult mouse β-cells using MafA transcriptional control sequences. Coexpression of MafB with MafA had no overt impact on mouse β-cells, suggesting that the human adult β-cell MAFA/MAFB heterodimer is functionally equivalent to the mouse MafA homodimer. However, MafB alone was unable to rescue the islet β-cell defects in a mouse mutant lacking MafA in β-cells. Of note, transgenic production of MafB in β-cells elevated tryptophan hydr...

Cell cycle (Georgetown, Tex.), 2017

Diabetes results from an inadequate mass of functional beta cells, due to either beta cell loss c... more Diabetes results from an inadequate mass of functional beta cells, due to either beta cell loss caused by autoimmune destruction (Type I diabetes) or beta cell failure in response to insulin resistance (Type II diabetes). Elucidating the mechanisms that regulate beta cell mass may be key to developing new techniques that foster beta cell regeneration as a cellular therapy to treat diabetes. While previous studies concluded that cyclin D2 is required for postnatal beta cell self-renewal in mice, it is not clear if cyclin D2 is sufficient to drive beta cell self-renewal. Using transgenic mice that overexpress cyclin D2 specifically in beta cells, we show that cyclin D2 overexpression increases beta cell self-renewal post-weaning and results in increased beta cell mass. Beta cells that overexpress cyclin D2 are responsive to glucose stimulation, suggesting they are functionally mature. Beta cells that overexpress cyclin D2 demonstrate an enhanced regenerative capacity after injury indu...

The Journal of clinical investigation, Jan 3, 2017

Pancreatic β cells have one of the highest protein secretion burdens in the body, as these cells ... more Pancreatic β cells have one of the highest protein secretion burdens in the body, as these cells must synthesize and secrete insulin in proportion to postprandial rises in blood glucose. Remarkably, it is now becoming clear that adult β cells retain plasticity and can dedifferentiate into embryonic fates or adopt alternate islet endocrine cell identities. This property is especially important, because changes in cell fate alter β cell function and could form the basis for defects in insulin secretion that occur early in the pathogenesis of the most prevalent form of β cell dysfunction, type 2 diabetes. In this issue, three different studies provide complementary perspectives on how the transcription factors NK2 homeobox 2 (NKX2.2), paired box 6 (PAX6), and LIM domain-binding protein 1 (LDB1) serve to maintain mature adult β cell identity, revealing clues as to how adult β cells can partially dedifferentiate or become reprogrammed into other islet endocrine cells.

Diabetes, 2016

Diabetes is associated with loss of functional pancreatic β-cells, and restoration of β-cells is ... more Diabetes is associated with loss of functional pancreatic β-cells, and restoration of β-cells is a major goal for regenerative therapies. Endogenous regeneration of β-cells via β-cell replication has the potential to restore cellular mass; however, pharmacological agents that promote regeneration or expansion of endogenous β-cells have been elusive. The regenerative capacity of β-cells declines rapidly with age, due to accumulation of p16INK4a, resulting in limited capacity for adult endocrine pancreas regeneration. Here, we show that transforming growth factor-β (TGF-β) signaling via Smad3 integrates with the trithorax complex to activate and maintain Ink4a expression to prevent β-cell replication. Importantly, inhibition of TGF-β signaling can result in repression of the Ink4a/Arf locus, resulting in increased β-cell replication in adult mice. Furthermore, small molecule inhibitors of the TGF-β pathway promote β-cell replication in human islets transplanted into NOD-scid IL-2Rgnul...

The Journal of clinical investigation, 2015

Pancreatic β cells secrete insulin in response to postprandial increases in glucose levels to pre... more Pancreatic β cells secrete insulin in response to postprandial increases in glucose levels to prevent hyperglycemia and inhibit insulin secretion under fasting conditions to protect against hypoglycemia. β cells lack this functional capability at birth and acquire glucose-stimulated insulin secretion (GSIS) during neonatal life. Here, we have shown that during postnatal life, the de novo DNA methyltransferase DNMT3A initiates a metabolic program by repressing key genes, thereby enabling the coupling of insulin secretion to glucose levels. In a murine model, β cell-specific deletion of Dnmt3a prevented the metabolic switch, resulting in loss of GSIS. DNMT3A bound to the promoters of the genes encoding hexokinase 1 (HK1) and lactate dehydrogenase A (LDHA) - both of which regulate the metabolic switch - and knockdown of these two key DNMT3A targets restored the GSIS response in islets from animals with β cell-specific Dnmt3a deletion. Furthermore, DNA methylation-mediated repression of...

Diabetes, 2006

Diabetes results from an inadequate mass of functional β-cells. Such inadequacy could result from... more Diabetes results from an inadequate mass of functional β-cells. Such inadequacy could result from loss of β-cells due to an immune assault or the inability to compensate for insulin resistance. Thus, mechanisms that regulate the number of β-cells will be key to understanding both the pathogenesis of diabetes and for developing therapies. In this study, we show that cell cycle regulator p27 plays a crucial role in establishing the number of β-cells formed before birth. We show that p27 accumulates in terminally differentiated β-cells during embryogenesis. Disabling p27 allows newly differentiated β-cells that are normally quiescent during embryogenesis to reenter the cell cycle and proliferate. As a consequence, excess β-cells are generated in the p27−/− mice, doubling their β-cell mass at birth. The early postnatal expansion of β-cell mass was unaffected in p27−/− mice, indicating that the main function of p27 is to maintain the quiescent state of newly differentiated β-cells genera...

Developmental Biology, 2006

In developing organs, the regulation of cell proliferation and cell cycle exit is coordinated. Ho... more In developing organs, the regulation of cell proliferation and cell cycle exit is coordinated. How this coordination is achieved, however, is not clear. We show that the cyclin kinase inhibitor p57 regulates cell cycle exit of progenitors during the early stages of pancreas formation. In the absence of p57, the number of cycling progenitors increases, although expansion of progenitor population is prevented by apoptosis. We report that p57 is a direct target of transcriptional repression by Notch effector, Hes1. Inactivation of Hes1 results in the upregulation of p57 expression in progenitors, leading to cell cycle arrest, precocious differentiation and depletion of the progenitor pool. We present evidence that in p57/Hes1 double null embryos, the absence of apoptosis results in the expansion of the progenitor population. We propose that Hes1 and p57 not only coordinate cell cycle exit and self-renewal of pancreatic progenitors during an early stage in organogenesis to regulate the number of pancreatic progenitors, but could also constitute a surveillance system to eliminate cells with aberrant cell cycle characteristics.

Journal of Clinical Investigation, 2013

Inadequate functional β cell mass underlies both type 1 and type 2 diabetes. β Cell growth and re... more Inadequate functional β cell mass underlies both type 1 and type 2 diabetes. β Cell growth and regeneration also decrease with age through mechanisms that are not fully understood. Age-dependent loss of enhancer of zeste homolog 2 (EZH2) prevents adult β cell replication through derepression of the gene encoding cyclindependent kinase inhibitor 2a (INK4a). We investigated whether replenishing EZH2 could reverse the agedependent increase of Ink4a transcription. We generated an inducible pancreatic β cell-specific Ezh2 transgenic mouse model and showed that transgene expression of Ezh2 was sufficient to increase β cell replication and regeneration in young adult mice. In mice older than 8 months, induction of Ezh2 was unable to repress Ink4a. Older mice had an enrichment of a trithorax group (TrxG) protein complex at the Ink4a locus. Knockdown of TrxG complex components, in conjunction with expression of Ezh2, resulted in Ink4a repression and increased replication of β cells in aged mice. These results indicate that combined modulation of polycomb group proteins, such as EZH2, along with TrxG proteins to repress Ink4a can rejuvenate the replication capacity of aged β cells. This study provides potential therapeutic targets for expansion of adult β cell mass. Conflict of interest: The authors have declared that no conflict of interest exists.

Development, 2001

The importance of mesenchymal-epithelial interactions for the proper development of the pancreas ... more The importance of mesenchymal-epithelial interactions for the proper development of the pancreas has been acknowledged since the early 1960s, even though the molecule(s) mediating this process have remained unknown. We demonstrate here that Fgf10, a member of the fibroblast growth factor family (FGFs), plays an essential role in this process. We show that Fgf10 is expressed in the mesenchyme directly adjacent to the early dorsal and ventral pancreatic epithelial buds. In Fgf10–/– mouse embryos, the evagination of the epithelium and the initial formation of the dorsal and ventral buds appear normal. However, the subsequent growth, differentiation and branching morphogenesis of the pancreatic epithelium are arrested; this is primarily due to a dramatic reduction in the proliferation of the epithelial progenitor cells marked by the production of the homeobox protein PDX1. Furthermore, FGF10 restores the population of PDX1-positive cells in organ cultures derived from Fgf10–/– embryos. ...

Blood, 2004

A debate has raged in recent years over whether cells within the bone marrow have the capacity to... more A debate has raged in recent years over whether cells within the bone marrow have the capacity to generate non-hematopoietic, specialized tissue in other organs. Previous reports have studied bone marrow transplantation in adult recipients; most models have found that tissue injury was necessary for organ engraftment of bone marrow and at best low levels of engraftment are seen. In the present study, we show complex ductal epithelial structures in the pancreas completely derived from bone marrow transplanted during the neonatal period in the absence of tissue injury. BRDU labeling showed that epithelial cells lining the ducts and islet cells were cycling at significantly higher levels in the neonatal than in the adult pancreas (P<0.0001). The contribution of transplanted bone marrow to neonatal pancreatic development was thus explored. To exclude the effect of injury on engraftment patterns, non-conditioned, immune deficient neonatal mice were used as bone marrow recipients. 5-10...

Nature clinical practice. Endocrinology & metabolism, 2007

Replication of beta cells is an important source of beta-cell expansion in early childhood. The r... more Replication of beta cells is an important source of beta-cell expansion in early childhood. The recent linkage of type 2 diabetes with several transcription factors involved in cell cycle regulation implies that growth of the beta-cell mass in early childhood might be an important determinant of risk for type 2 diabetes. Under some circumstances, including obesity and pregnancy, the beta-cell mass is adaptively increased in adult humans. The mechanisms by which this adaptive growth occurs and the relative contributions of beta-cell replication or of mechanisms independent of beta-cell replication are unknown. Also, although there is interest in the potential for beta-cell regeneration as a therapeutic approach in both type 1 and 2 diabetes, little is yet known about the potential sources of new beta cells in adult humans. In common with other cell types, replicating beta cells have an increased vulnerability to apoptosis, which is likely to limit the therapeutic value of inducing be...

Stem Cells, 2006

Recent reports suggest that bone marrow–derived cells engraft and differentiate into pancreatic t... more Recent reports suggest that bone marrow–derived cells engraft and differentiate into pancreatic tissue at very low frequency after pancreatic injury. All such studies have used adult recipients. The aim of our studies was to investigate the potential of bone marrow to contribute to the exocrine and endocrine components of the pancreas during the normal rapid growth of the organ that occurs during the neonatal period. Five to ten million bone marrow cells from adult, male, transgenic, green fluorescent protein (GFP) mice were injected into neonatal nonobese diabetic/severely compromised immunodeficient/β2microglobulin-null mice 24 hours after birth. Two months after bone marrow transplantation, pancreas tissue was analyzed with fluorescence immunohistochemistry and fluorescence in situ hybridization (FISH). Co-staining of GFP, with anticytokeratin antibody, and with FISH for the presence of donor Y chromosome indicated that up to 40% of ducts (median 4.6%) contained epithelial cells ...

Proceedings of the National Academy of Sciences, 1997

Smad proteins are critical intracellular mediators of signaling by growth and differentiation fac... more Smad proteins are critical intracellular mediators of signaling by growth and differentiation factors of the transforming growth factor β superfamily. We have isolated a member of the Smad family, Smad8, from a rat brain cDNA library and biochemically and functionally characterized its ability to transduce signals from serine kinase receptors. In Xenopus embryo, Smad8 is able to transcriptionally activate a subset of mesoderm target genes similar to those induced by the receptor serine kinase, activin receptor-like kinase (ALK)-2. Smad8 can be specifically phosphorylated by a constitutively active ALK-2 but not the related receptor serine kinase, ALK-4. In response to signaling from ALK-2, Smad8 associates with a common regulatory molecule, Smad4, and this association leads to a synergistic effect on gene transcription. Furthermore, Smad8 is able to rescue the expression of mesoderm genes blocked by truncated ALK-2 in the embryo. These results indicate that Smad8 can function as a d...

PLoS ONE, 2009

Background Apoptosis is a hallmark of β-cell death in both type 1 and type 2 diabetes mellitus. U... more Background Apoptosis is a hallmark of β-cell death in both type 1 and type 2 diabetes mellitus. Understanding how apoptosis contributes to β-cell turnover may lead to strategies to prevent progression of diabetes. A key mediator of apoptosis, mitochondrial function, and cell survival is apoptosis inducing factor (AIF). In the present study, we investigated the role of AIF on β-cell mass and survival using the Harlequin (Hq) mutant mice, which are hypomorphic for AIF. Methodology/Principal Findings Immunohistochemical evaluation of pancreata from Hq mutant mice displayed much smaller islets compared to wild-type mice (WT). Analysis of β-cell mass in these mice revealed a greater than 4-fold reduction in β-cell mass together with an 8-fold increase in β-cell apoptosis. Analysis of cell cycle dynamics, using BrdU pulse as a marker for cells in S-phase, did not detect significant differences in the frequency of β-cells in S-phase. In contrast, double staining for phosphorylated Histone ...

OBJECTIVEType 1 Diabetes (T1D) results from progressive loss of pancreatic beta cells due to auto... more OBJECTIVEType 1 Diabetes (T1D) results from progressive loss of pancreatic beta cells due to autoimmune destruction. We recently reported that during the natural history of T1D in humans and the female nonobese diabetic (NOD) mouse model, beta cells acquire a senescence-associated secretory phenotype (SASP) that is a major driver of disease onset and progression, but the mechanisms that activate SASP in beta cells were not explored. The objective of this study was to identify transcriptional mechanisms of SASP activation in beta cells.METHODSWe used the female NOD mouse model of spontaneous autoimmune T1D and ex vivo experiments on NOD mouse and human islets to test the hypothesis that Bromodomain Extra-Terminal domain (BET) proteins activate the beta cell SASP transcriptional program.RESULTSHere we show that beta cell SASP is transcriptionally controlled by BET proteins, including BRD4. Chromatin analysis of key beta cell SASP genes in NOD islets revealed binding of BRD4 at active ...

Journal of Clinical Investigation, 2019

Nature Cell Biology, 2019

In the version of this article originally published, the Gene Expression Omnibus (GEO) accession ... more In the version of this article originally published, the Gene Expression Omnibus (GEO) accession number listed in the data availability section was incorrectly given as GSE10979 instead of GSE109795. The sentence should read "RNA-seq data that support the findings of this study have been deposited in the Gene Expression Omnibus (GEO) under accession code GSE109795, " and the code should link to https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE109795. The error has been corrected in the HTML and PDF versions of the paper.

Nature Cell Biology, 2019

Despite advances in the differentiation of insulin-producing cells from human embryonic stem cell... more Despite advances in the differentiation of insulin-producing cells from human embryonic stem cells, the generation of mature functional β cells in vitro has remained elusive. To accomplish this goal, we have developed cell culture conditions to closely mimic events occurring during pancreatic islet organogenesis and β cell maturation. In particular, we have focused on recapitulating endocrine cell clustering by isolating and reaggregating immature β-like cells to form islet-sized enriched β-clusters (eBCs). eBCs display physiological properties analogous to primary human β cells, including robust dynamic insulin secretion, increased calcium signalling in response to secretagogues, and improved mitochondrial energization. Notably, endocrine cell *

Diabetes, 2018

The sustained expression of the MAFB transcription factor in human islet β-cells represents a dis... more The sustained expression of the MAFB transcription factor in human islet β-cells represents a distinct difference in mice. Moreover, mRNA expression of closely related and islet β-cell–enriched MAFA does not peak in humans until after 9 years of age. We show that the MAFA protein also is weakly produced within the juvenile human islet β-cell population and that MafB expression is postnatally restricted in mouse β-cells by de novo DNA methylation. To gain insight into how MAFB affects human β-cells, we developed a mouse model to ectopically express MafB in adult mouse β-cells using MafA transcriptional control sequences. Coexpression of MafB with MafA had no overt impact on mouse β-cells, suggesting that the human adult β-cell MAFA/MAFB heterodimer is functionally equivalent to the mouse MafA homodimer. However, MafB alone was unable to rescue the islet β-cell defects in a mouse mutant lacking MafA in β-cells. Of note, transgenic production of MafB in β-cells elevated tryptophan hydr...

Cell cycle (Georgetown, Tex.), 2017

Diabetes results from an inadequate mass of functional beta cells, due to either beta cell loss c... more Diabetes results from an inadequate mass of functional beta cells, due to either beta cell loss caused by autoimmune destruction (Type I diabetes) or beta cell failure in response to insulin resistance (Type II diabetes). Elucidating the mechanisms that regulate beta cell mass may be key to developing new techniques that foster beta cell regeneration as a cellular therapy to treat diabetes. While previous studies concluded that cyclin D2 is required for postnatal beta cell self-renewal in mice, it is not clear if cyclin D2 is sufficient to drive beta cell self-renewal. Using transgenic mice that overexpress cyclin D2 specifically in beta cells, we show that cyclin D2 overexpression increases beta cell self-renewal post-weaning and results in increased beta cell mass. Beta cells that overexpress cyclin D2 are responsive to glucose stimulation, suggesting they are functionally mature. Beta cells that overexpress cyclin D2 demonstrate an enhanced regenerative capacity after injury indu...

The Journal of clinical investigation, Jan 3, 2017

Pancreatic β cells have one of the highest protein secretion burdens in the body, as these cells ... more Pancreatic β cells have one of the highest protein secretion burdens in the body, as these cells must synthesize and secrete insulin in proportion to postprandial rises in blood glucose. Remarkably, it is now becoming clear that adult β cells retain plasticity and can dedifferentiate into embryonic fates or adopt alternate islet endocrine cell identities. This property is especially important, because changes in cell fate alter β cell function and could form the basis for defects in insulin secretion that occur early in the pathogenesis of the most prevalent form of β cell dysfunction, type 2 diabetes. In this issue, three different studies provide complementary perspectives on how the transcription factors NK2 homeobox 2 (NKX2.2), paired box 6 (PAX6), and LIM domain-binding protein 1 (LDB1) serve to maintain mature adult β cell identity, revealing clues as to how adult β cells can partially dedifferentiate or become reprogrammed into other islet endocrine cells.

Diabetes, 2016

Diabetes is associated with loss of functional pancreatic β-cells, and restoration of β-cells is ... more Diabetes is associated with loss of functional pancreatic β-cells, and restoration of β-cells is a major goal for regenerative therapies. Endogenous regeneration of β-cells via β-cell replication has the potential to restore cellular mass; however, pharmacological agents that promote regeneration or expansion of endogenous β-cells have been elusive. The regenerative capacity of β-cells declines rapidly with age, due to accumulation of p16INK4a, resulting in limited capacity for adult endocrine pancreas regeneration. Here, we show that transforming growth factor-β (TGF-β) signaling via Smad3 integrates with the trithorax complex to activate and maintain Ink4a expression to prevent β-cell replication. Importantly, inhibition of TGF-β signaling can result in repression of the Ink4a/Arf locus, resulting in increased β-cell replication in adult mice. Furthermore, small molecule inhibitors of the TGF-β pathway promote β-cell replication in human islets transplanted into NOD-scid IL-2Rgnul...

The Journal of clinical investigation, 2015

Pancreatic β cells secrete insulin in response to postprandial increases in glucose levels to pre... more Pancreatic β cells secrete insulin in response to postprandial increases in glucose levels to prevent hyperglycemia and inhibit insulin secretion under fasting conditions to protect against hypoglycemia. β cells lack this functional capability at birth and acquire glucose-stimulated insulin secretion (GSIS) during neonatal life. Here, we have shown that during postnatal life, the de novo DNA methyltransferase DNMT3A initiates a metabolic program by repressing key genes, thereby enabling the coupling of insulin secretion to glucose levels. In a murine model, β cell-specific deletion of Dnmt3a prevented the metabolic switch, resulting in loss of GSIS. DNMT3A bound to the promoters of the genes encoding hexokinase 1 (HK1) and lactate dehydrogenase A (LDHA) - both of which regulate the metabolic switch - and knockdown of these two key DNMT3A targets restored the GSIS response in islets from animals with β cell-specific Dnmt3a deletion. Furthermore, DNA methylation-mediated repression of...

Diabetes, 2006

Diabetes results from an inadequate mass of functional β-cells. Such inadequacy could result from... more Diabetes results from an inadequate mass of functional β-cells. Such inadequacy could result from loss of β-cells due to an immune assault or the inability to compensate for insulin resistance. Thus, mechanisms that regulate the number of β-cells will be key to understanding both the pathogenesis of diabetes and for developing therapies. In this study, we show that cell cycle regulator p27 plays a crucial role in establishing the number of β-cells formed before birth. We show that p27 accumulates in terminally differentiated β-cells during embryogenesis. Disabling p27 allows newly differentiated β-cells that are normally quiescent during embryogenesis to reenter the cell cycle and proliferate. As a consequence, excess β-cells are generated in the p27−/− mice, doubling their β-cell mass at birth. The early postnatal expansion of β-cell mass was unaffected in p27−/− mice, indicating that the main function of p27 is to maintain the quiescent state of newly differentiated β-cells genera...

Developmental Biology, 2006

In developing organs, the regulation of cell proliferation and cell cycle exit is coordinated. Ho... more In developing organs, the regulation of cell proliferation and cell cycle exit is coordinated. How this coordination is achieved, however, is not clear. We show that the cyclin kinase inhibitor p57 regulates cell cycle exit of progenitors during the early stages of pancreas formation. In the absence of p57, the number of cycling progenitors increases, although expansion of progenitor population is prevented by apoptosis. We report that p57 is a direct target of transcriptional repression by Notch effector, Hes1. Inactivation of Hes1 results in the upregulation of p57 expression in progenitors, leading to cell cycle arrest, precocious differentiation and depletion of the progenitor pool. We present evidence that in p57/Hes1 double null embryos, the absence of apoptosis results in the expansion of the progenitor population. We propose that Hes1 and p57 not only coordinate cell cycle exit and self-renewal of pancreatic progenitors during an early stage in organogenesis to regulate the number of pancreatic progenitors, but could also constitute a surveillance system to eliminate cells with aberrant cell cycle characteristics.

Journal of Clinical Investigation, 2013

Inadequate functional β cell mass underlies both type 1 and type 2 diabetes. β Cell growth and re... more Inadequate functional β cell mass underlies both type 1 and type 2 diabetes. β Cell growth and regeneration also decrease with age through mechanisms that are not fully understood. Age-dependent loss of enhancer of zeste homolog 2 (EZH2) prevents adult β cell replication through derepression of the gene encoding cyclindependent kinase inhibitor 2a (INK4a). We investigated whether replenishing EZH2 could reverse the agedependent increase of Ink4a transcription. We generated an inducible pancreatic β cell-specific Ezh2 transgenic mouse model and showed that transgene expression of Ezh2 was sufficient to increase β cell replication and regeneration in young adult mice. In mice older than 8 months, induction of Ezh2 was unable to repress Ink4a. Older mice had an enrichment of a trithorax group (TrxG) protein complex at the Ink4a locus. Knockdown of TrxG complex components, in conjunction with expression of Ezh2, resulted in Ink4a repression and increased replication of β cells in aged mice. These results indicate that combined modulation of polycomb group proteins, such as EZH2, along with TrxG proteins to repress Ink4a can rejuvenate the replication capacity of aged β cells. This study provides potential therapeutic targets for expansion of adult β cell mass. Conflict of interest: The authors have declared that no conflict of interest exists.

Development, 2001

The importance of mesenchymal-epithelial interactions for the proper development of the pancreas ... more The importance of mesenchymal-epithelial interactions for the proper development of the pancreas has been acknowledged since the early 1960s, even though the molecule(s) mediating this process have remained unknown. We demonstrate here that Fgf10, a member of the fibroblast growth factor family (FGFs), plays an essential role in this process. We show that Fgf10 is expressed in the mesenchyme directly adjacent to the early dorsal and ventral pancreatic epithelial buds. In Fgf10–/– mouse embryos, the evagination of the epithelium and the initial formation of the dorsal and ventral buds appear normal. However, the subsequent growth, differentiation and branching morphogenesis of the pancreatic epithelium are arrested; this is primarily due to a dramatic reduction in the proliferation of the epithelial progenitor cells marked by the production of the homeobox protein PDX1. Furthermore, FGF10 restores the population of PDX1-positive cells in organ cultures derived from Fgf10–/– embryos. ...

Blood, 2004

A debate has raged in recent years over whether cells within the bone marrow have the capacity to... more A debate has raged in recent years over whether cells within the bone marrow have the capacity to generate non-hematopoietic, specialized tissue in other organs. Previous reports have studied bone marrow transplantation in adult recipients; most models have found that tissue injury was necessary for organ engraftment of bone marrow and at best low levels of engraftment are seen. In the present study, we show complex ductal epithelial structures in the pancreas completely derived from bone marrow transplanted during the neonatal period in the absence of tissue injury. BRDU labeling showed that epithelial cells lining the ducts and islet cells were cycling at significantly higher levels in the neonatal than in the adult pancreas (P<0.0001). The contribution of transplanted bone marrow to neonatal pancreatic development was thus explored. To exclude the effect of injury on engraftment patterns, non-conditioned, immune deficient neonatal mice were used as bone marrow recipients. 5-10...

Nature clinical practice. Endocrinology & metabolism, 2007

Replication of beta cells is an important source of beta-cell expansion in early childhood. The r... more Replication of beta cells is an important source of beta-cell expansion in early childhood. The recent linkage of type 2 diabetes with several transcription factors involved in cell cycle regulation implies that growth of the beta-cell mass in early childhood might be an important determinant of risk for type 2 diabetes. Under some circumstances, including obesity and pregnancy, the beta-cell mass is adaptively increased in adult humans. The mechanisms by which this adaptive growth occurs and the relative contributions of beta-cell replication or of mechanisms independent of beta-cell replication are unknown. Also, although there is interest in the potential for beta-cell regeneration as a therapeutic approach in both type 1 and 2 diabetes, little is yet known about the potential sources of new beta cells in adult humans. In common with other cell types, replicating beta cells have an increased vulnerability to apoptosis, which is likely to limit the therapeutic value of inducing be...

Stem Cells, 2006

Recent reports suggest that bone marrow–derived cells engraft and differentiate into pancreatic t... more Recent reports suggest that bone marrow–derived cells engraft and differentiate into pancreatic tissue at very low frequency after pancreatic injury. All such studies have used adult recipients. The aim of our studies was to investigate the potential of bone marrow to contribute to the exocrine and endocrine components of the pancreas during the normal rapid growth of the organ that occurs during the neonatal period. Five to ten million bone marrow cells from adult, male, transgenic, green fluorescent protein (GFP) mice were injected into neonatal nonobese diabetic/severely compromised immunodeficient/β2microglobulin-null mice 24 hours after birth. Two months after bone marrow transplantation, pancreas tissue was analyzed with fluorescence immunohistochemistry and fluorescence in situ hybridization (FISH). Co-staining of GFP, with anticytokeratin antibody, and with FISH for the presence of donor Y chromosome indicated that up to 40% of ducts (median 4.6%) contained epithelial cells ...

Proceedings of the National Academy of Sciences, 1997

Smad proteins are critical intracellular mediators of signaling by growth and differentiation fac... more Smad proteins are critical intracellular mediators of signaling by growth and differentiation factors of the transforming growth factor β superfamily. We have isolated a member of the Smad family, Smad8, from a rat brain cDNA library and biochemically and functionally characterized its ability to transduce signals from serine kinase receptors. In Xenopus embryo, Smad8 is able to transcriptionally activate a subset of mesoderm target genes similar to those induced by the receptor serine kinase, activin receptor-like kinase (ALK)-2. Smad8 can be specifically phosphorylated by a constitutively active ALK-2 but not the related receptor serine kinase, ALK-4. In response to signaling from ALK-2, Smad8 associates with a common regulatory molecule, Smad4, and this association leads to a synergistic effect on gene transcription. Furthermore, Smad8 is able to rescue the expression of mesoderm genes blocked by truncated ALK-2 in the embryo. These results indicate that Smad8 can function as a d...

PLoS ONE, 2009

Background Apoptosis is a hallmark of β-cell death in both type 1 and type 2 diabetes mellitus. U... more Background Apoptosis is a hallmark of β-cell death in both type 1 and type 2 diabetes mellitus. Understanding how apoptosis contributes to β-cell turnover may lead to strategies to prevent progression of diabetes. A key mediator of apoptosis, mitochondrial function, and cell survival is apoptosis inducing factor (AIF). In the present study, we investigated the role of AIF on β-cell mass and survival using the Harlequin (Hq) mutant mice, which are hypomorphic for AIF. Methodology/Principal Findings Immunohistochemical evaluation of pancreata from Hq mutant mice displayed much smaller islets compared to wild-type mice (WT). Analysis of β-cell mass in these mice revealed a greater than 4-fold reduction in β-cell mass together with an 8-fold increase in β-cell apoptosis. Analysis of cell cycle dynamics, using BrdU pulse as a marker for cells in S-phase, did not detect significant differences in the frequency of β-cells in S-phase. In contrast, double staining for phosphorylated Histone ...