RESUMO
Mutations in CDKN1C, encoding p57KIP2, a canonical cell cycle inhibitor, underlie multiple pediatric endocrine syndromes. Despite this central role in disease, little is known about the structure and function of p57KIP2 in the human pancreatic beta cell. Since p57KIP2 is predominantly nuclear in human beta cells, we hypothesized that disease-causing mutations in its nuclear localization sequence (NLS) may correlate with abnormal phenotypes. We prepared RIP1 insulin promoter-driven adenoviruses encoding deletions of multiple disease-associated but unexplored regions of p57KIP2 and performed a comprehensive structure-function analysis of CDKN1C/p57KIP2. Real-time polymerase chain reaction and immunoblot analyses confirmed p57KIP2 overexpression, construct size, and beta cell specificity. By immunocytochemistry, wild-type (WT) p57KIP2 displayed nuclear localization. In contrast, deletion of a putative NLS at amino acids 278-281 failed to access the nucleus. Unexpectedly, we identified a second downstream NLS at amino acids 312-316. Further analysis showed that each individual NLS is required for nuclear localization, but neither alone is sufficient. In summary, p57KIP2 contains a classical bipartite NLS characterized by 2 clusters of positively charged amino acids separated by a proline-rich linker region. Variants in the sequences encoding these 2 NLS sequences account for functional p57KIP2 loss and beta cell expansion seen in human disease.
Assuntos
Inibidor de Quinase Dependente de Ciclina p57 , Células Secretoras de Insulina , Sinais de Localização Nuclear , Humanos , Sequência de Aminoácidos , Aminoácidos/metabolismo , Núcleo Celular/metabolismo , Células Secretoras de Insulina/metabolismo , Sinais de Localização Nuclear/genética , Sinais de Localização Nuclear/metabolismo , Inibidor de Quinase Dependente de Ciclina p57/genéticaRESUMO
Resistance to regeneration of insulin-producing pancreatic ß cells is a fundamental challenge for type 1 and type 2 diabetes. Recently, small molecule inhibitors of the kinase DYRK1A have proven effective in inducing adult human ß cells to proliferate, but their detailed mechanism of action is incompletely understood. We interrogated our human insulinoma and ß cell transcriptomic databases seeking to understand why ß cells in insulinomas proliferate, while normal ß cells do not. This search reveals the DREAM complex as a central regulator of quiescence in human ß cells. The DREAM complex consists of a module of transcriptionally repressive proteins that assemble in response to DYRK1A kinase activity, thereby inducing and maintaining cellular quiescence. In the absence of DYRK1A, DREAM subunits reassemble into the pro-proliferative MMB complex. Here, we demonstrate that small molecule DYRK1A inhibitors induce human ß cells to replicate by converting the repressive DREAM complex to its pro-proliferative MMB conformation.
Assuntos
Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Insulinoma , Neoplasias Pancreáticas , Adulto , Proliferação de Células , Humanos , Células Secretoras de Insulina/metabolismo , Insulinoma/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/metabolismoRESUMO
A quantitative deficiency of normally functioning insulin-producing pancreatic beta cells is a major contributor to all common forms of diabetes. This is the underlying premise for attempts to replace beta cells in people with diabetes by pancreas transplantation, pancreatic islet transplantation, and transplantation of beta cells or pancreatic islets derived from human stem cells. While progress is rapid and impressive in the beta cell replacement field, these approaches are expensive, and for transplant approaches, limited by donor organ availability. For these reasons, beta cell replacement will not likely become available to the hundreds of millions of people around the world with diabetes. Since the large majority of people with diabetes have some residual beta cells in their pancreata, an alternate approach to reversing diabetes would be developing pharmacologic approaches to induce these residual beta cells to regenerate and expand in a way that also permits normal function. Unfortunately, despite the broad availability of multiple classes of diabetes drugs in the current diabetes armamentarium, none has the ability to induce regeneration or expansion of human beta cells. Development of such drugs would be transformative for diabetes care around the world. This picture has begun to change. Over the past half-decade, a novel class of beta cell regenerative small molecules has emerged: the DYRK1A inhibitors. Their emergence has tremendous potential, but many areas of uncertainty and challenge remain. In this review, we summarize the accomplishments in the world of beta cell regenerative drug development and summarize areas in which most experts would agree. We also outline and summarize areas of disagreement or lack of unanimity, of controversy in the field, of obstacles to beta cell regeneration, and of challenges that will need to be overcome in order to establish human beta cell regenerative drug therapeutics as a clinically viable class of diabetes drugs.
Assuntos
Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Células Secretoras de Insulina/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Regeneração/efeitos dos fármacos , Diabetes Mellitus Tipo 1/fisiopatologia , Diabetes Mellitus Tipo 2/fisiopatologia , Humanos , Células Secretoras de Insulina/citologia , Quinases DyrkRESUMO
Human insulinomas are rare, benign, slowly proliferating, insulin-producing beta cell tumors that provide a molecular "recipe" or "roadmap" for pathways that control human beta cell regeneration. An earlier study revealed abnormal methylation in the imprinted p15.5-p15.4 region of chromosome 11, known to be abnormally methylated in another disorder of expanded beta cell mass and function: the focal variant of congenital hyperinsulinism. Here, we compare deep DNA methylome sequencing on 19 human insulinomas, and five sets of normal beta cells. We find a remarkably consistent, abnormal methylation pattern in insulinomas. The findings suggest that abnormal insulin (INS) promoter methylation and altered transcription factor expression create alternative drivers of INS expression, replacing canonical PDX1-driven beta cell specification with a pathological, looping, distal enhancer-based form of transcriptional regulation. Finally, NFaT transcription factors, rather than the canonical PDX1 enhancer complex, are predicted to drive INS transactivation.
Assuntos
Regulação Neoplásica da Expressão Gênica , Insulina/genética , Insulina/metabolismo , Insulinoma/genética , Insulinoma/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Adulto , Idoso , Sítios de Ligação , Biologia Computacional , Metilação de DNA , Feminino , Proteínas de Homeodomínio/metabolismo , Humanos , Células Secretoras de Insulina/metabolismo , Masculino , Doenças Metabólicas/genética , Doenças Metabólicas/metabolismo , Pessoa de Meia-Idade , Regiões Promotoras Genéticas , Transativadores/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Adulto JovemRESUMO
Small molecule inhibitors of dual specificity, tyrosine phosphorylation-regulated kinase 1A (DYRK1A), including harmine and others, are able to drive human ß cell regeneration. While DYRK1A is certainly a target of this class, whether it is the only or the most important target is uncertain. Here, we employ a combined pharmacologic and genetic approach to refine the potential mitogenic targets of the DYRK1A inhibitor family in human islets. A combination of human ß cell RNA sequencing, DYRK1A inhibitor kinome screens, pharmacologic inhibitors, and targeted silencing of candidate genes confirms that DYRK1A is a central target. Surprisingly, however, DYRK1B also proves to be an important target: silencing DYRK1A results in an increase in DYRK1B. Simultaneous silencing of both DYRK1A and DYRK1B yields greater ß cell proliferation than silencing either individually. Importantly, other potential kinases, such as the CLK and the GSK3 families, are excluded as important harmine targets. Finally, we describe adenoviruses that are able to silence up to 7 targets simultaneously. Collectively, we report that inhibition of both DYRK1A and DYRK1B is required for induction of maximal rates of human ß cell proliferation, and we provide clarity for future efforts in structure-based drug design for human ß cell regenerative drugs.
Assuntos
Células Secretoras de Insulina/metabolismo , Mitógenos/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/efeitos dos fármacos , Proteínas Tirosina Quinases/efeitos dos fármacos , Adolescente , Adulto , Idoso , Proliferação de Células/efeitos dos fármacos , Feminino , Expressão Gênica , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Harmina/metabolismo , Harmina/farmacologia , Humanos , Insulinoma , Masculino , Pessoa de Meia-Idade , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Adulto Jovem , Quinases DyrkRESUMO
Type 1 diabetes (T1D) is caused by autoimmune destruction of pancreatic ß cells. Mounting evidence supports a central role for ß cell alterations in triggering the activation of self-reactive T cells in T1D. However, the early deleterious events that occur in ß cells, underpinning islet autoimmunity, are not known. We hypothesized that epigenetic modifications induced in ß cells by inflammatory mediators play a key role in initiating the autoimmune response. We analyzed DNA methylation (DNAm) patterns and gene expression in human islets exposed to IFN-α, a cytokine associated with T1D development. We found that IFN-α triggers DNA demethylation and increases expression of genes controlling inflammatory and immune pathways. We then demonstrated that DNA demethylation was caused by upregulation of the exoribonuclease, PNPase old-35 (PNPT1), which caused degradation of miR-26a. This in turn promoted the upregulation of ten-eleven translocation 2 (TET2) enzyme and increased 5-hydroxymethylcytosine levels in human islets and pancreatic ß cells. Moreover, we showed that specific IFN-α expression in the ß cells of IFNα-INS1CreERT2 transgenic mice led to development of T1D that was preceded by increased islet DNA hydroxymethylation through a PNPT1/TET2-dependent mechanism. Our results suggest a new mechanism through which IFN-α regulates DNAm in ß cells, leading to changes in expression of genes in inflammatory and immune pathways that can initiate islet autoimmunity in T1D.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Epigênese Genética , Exorribonucleases/metabolismo , Células Secretoras de Insulina/metabolismo , Interferon-alfa/metabolismo , MicroRNAs/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Animais , Linhagem Celular , Citocinas/metabolismo , Metilação de DNA , Proteínas de Ligação a DNA/genética , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Dioxigenases , Exorribonucleases/genética , Feminino , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos Transgênicos , MicroRNAs/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Proto-Oncogênicas/genética , Linfócitos T/metabolismo , Regulação para CimaRESUMO
Small-molecule inhibitors of dual-specificity tyrosine-regulated kinase 1A (DYRK1A) induce human beta cells to proliferate, generating a labeling index of 1.5%-3%. Here, we demonstrate that combined pharmacologic inhibition of DYRK1A and transforming growth factor beta superfamily (TGFßSF)/SMAD signaling generates remarkable further synergistic increases in human beta cell proliferation (average labeling index, 5%-8%, and as high as 15%-18%), and increases in both mouse and human beta cell numbers. This synergy reflects activation of cyclins and cdks by DYRK1A inhibition, accompanied by simultaneous reductions in key cell-cycle inhibitors (CDKN1C and CDKN1A). The latter results from interference with the basal Trithorax- and SMAD-mediated transactivation of CDKN1C and CDKN1A. Notably, combined DYRK1A and TGFß inhibition allows preservation of beta cell differentiated function. These beneficial effects extend from normal human beta cells and stem cell-derived human beta cells to those from people with type 2 diabetes, and occur both in vitro and in vivo.
Assuntos
Diabetes Mellitus Tipo 2 , Harmina/farmacologia , Células Secretoras de Insulina , Inibidores da Monoaminoxidase/farmacologia , Fator de Crescimento Transformador beta/antagonistas & inibidores , Adolescente , Adulto , Idoso , Animais , Linhagem Celular , Proliferação de Células , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Feminino , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Proteína de Leucina Linfoide-Mieloide/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Smad/antagonistas & inibidores , Células-Tronco , Adulto Jovem , Quinases DyrkRESUMO
Although diabetes results in part from a deficiency of normal pancreatic beta cells, inducing human beta cells to regenerate is difficult. Reasoning that insulinomas hold the "genomic recipe" for beta cell expansion, we surveyed 38 human insulinomas to obtain insights into therapeutic pathways for beta cell regeneration. An integrative analysis of whole-exome and RNA-sequencing data was employed to extensively characterize the genomic and molecular landscape of insulinomas relative to normal beta cells. Here, we show at the pathway level that the majority of the insulinomas display mutations, copy number variants and/or dysregulation of epigenetic modifying genes, most prominently in the polycomb and trithorax families. Importantly, these processes are coupled to co-expression network modules associated with cell proliferation, revealing candidates for inducing beta cell regeneration. Validation of key computational predictions supports the concept that understanding the molecular complexity of insulinoma may be a valuable approach to diabetes drug discovery.Diabetes results in part from a deficiency of functional pancreatic beta cells. Here, the authors study the genomic and epigenetic landscapes of human insulinomas to gain insight into possible pathways for therapeutic beta cell regeneration, highlighting epigenetic genes and pathways.
Assuntos
Proliferação de Células/genética , Diabetes Mellitus Tipo 1/terapia , Células Secretoras de Insulina/metabolismo , Insulinoma/genética , Neoplasias Pancreáticas/genética , Regeneração/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Diabetes Mellitus Tipo 1/metabolismo , Epigênese Genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Insulinoma/metabolismo , Masculino , Pessoa de Meia-Idade , Neoplasias Pancreáticas/metabolismoRESUMO
PINCH-1 is a LIM-only domain protein that forms a ternary complex with integrin-linked kinase (ILK) and parvin (to form the IPP complex) downstream of integrins. Here, we demonstrate that PINCH-1 (also known as Lims1) gene ablation in the epidermis of mice caused epidermal detachment from the basement membrane, epidermal hyperthickening and progressive hair loss. PINCH-1-deficient keratinocytes also displayed profound adhesion, spreading and migration defects in vitro that were substantially more severe than those of ILK-deficient keratinocytes indicating that PINCH-1 also exerts functions in an ILK-independent manner. By isolating the PINCH-1 interactome, the LIM-domain-containing and actin-binding protein epithelial protein lost in neoplasm (EPLIN, also known as LIMA1) was identified as a new PINCH-1-associated protein. EPLIN localized, in a PINCH-1-dependent manner, to integrin adhesion sites of keratinocytes in vivo and in vitro and its depletion severely attenuated keratinocyte spreading and migration on collagen and fibronectin without affecting PINCH-1 levels in focal adhesions. Given that the low PINCH-1 levels in ILK-deficient keratinocytes were sufficient to recruit EPLIN to integrin adhesions, our findings suggest that PINCH-1 regulates integrin-mediated adhesion of keratinocytes through the interactions with ILK as well as EPLIN.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Movimento Celular/fisiologia , Proteínas do Citoesqueleto/metabolismo , Adesões Focais/metabolismo , Integrinas/metabolismo , Queratinócitos/metabolismo , Proteínas com Domínio LIM/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Células Cultivadas , Proteínas do Citoesqueleto/genética , Adesões Focais/genética , Integrinas/genética , Queratinócitos/citologia , Proteínas com Domínio LIM/genética , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos TransgênicosRESUMO
The focal adhesion (FA) protein PINCH-1 is required for the survival of primitive endoderm (PrE) cells. How PINCH-1 regulates this fundamental process is not known. Here, we use embryoid bodies (EBs) and isolated EB-derived PrE cells to investigate the mechanisms by which PINCH-1 promotes PrE survival. We report that loss of PINCH-1 in PrE cells leads to a sustained activity of JNK and the pro-apoptotic factor Bax. Mechanistically, the sustained JNK activation was due to diminished levels of the JNK inhibitory factor Ras suppressor protein-1 (RSU-1), whose stability was severely reduced upon loss of PINCH-1. Chemical inhibition of JNK attenuated apoptosis of PrE cells but failed to reduce Bax activity. The increased Bax activity was associated with reduced integrin signalling and diminished Bcl-2 levels, which were shown to inhibit Bax. Altogether our findings show that PINCH-1 is a pro-survival factor that prevents apoptosis of PrE cells by modulating two independent signalling pathways; PINCH-1 inhibits JNK-mediated apoptosis by stabilising the PINCH-1 binding protein RSU-1 and promotes Bcl-2-dependent pro-survival signalling downstream of integrins.