A smad signaling network regulates islet cell proliferation.

Pancreatic ß-cell loss and dysfunction are critical components of all types of diabetes. Human and rodent ß-cells are able to proliferate, and this proliferation is an important defense against the evolution and progression of diabetes. Transforming growth factor-ß (TGF-ß) signaling has been shown to affect ß-cell development, proliferation, and function, but ß-cell proliferation is thought to be the only source of new ß-cells in the adult. Recently, ß-cell dedifferentiation has been shown to be an important contributory mechanism to ß-cell failure. In this study, we tie together these two pathways by showing that a network of intracellular TGF-ß regulators, smads 7, 2, and 3, control ß-cell proliferation after ß-cell loss, and specifically, smad7 is necessary for that ß-cell proliferation. Importantly, this smad7-mediated proliferation appears to entail passing through a transient, nonpathologic dedifferentiation of ß-cells to a pancreatic polypeptide-fold hormone-positive state. TGF-ß receptor II appears to be a receptor important for controlling the status of the smad network in ß-cells. These studies should help our understanding of properly regulated ß-cell replication.

No evidence for ß cell neogenesis in murine adult pancreas.

Whether facultative ß cell progenitors exist in the adult pancreas is a major unsolved question. To date, lineage-tracing studies have provided conflicting results. To track ß cell neogenesis in vivo, we generated transgenic mice that transiently coexpress mTomato and GFP in a time-sensitive, nonconditional Cre-mediated manner, so that insulin-producing cells express GFP under control of the insulin promoter, while all other cells express mTomato (INSCremTmG mice). Newly differentiated ß cells were detected by flow cytometry and fluorescence microscopy, taking advantage of their transient coexpression of GFP and mTomato fluorescent proteins. We found that ß cell neogenesis predominantly occurs during embryogenesis, decreases dramatically shortly after birth, and is completely absent in adults across various models of ß cell loss, ß cell growth and regeneration, and inflammation. Moreover, we demonstrated upregulation of neurogenin 3 (NGN3) in both proliferating ducts and preexisting ß cells in the ligated pancreatic tail after pancreatic ductal ligation. These results are consistent with some recent reports, but argue against the widely held belief that NGN3 marks cells undergoing endocrine neogenesis in the pancreas. Our data suggest that ß cell neogenesis in the adult pancreas occurs rarely, if ever, under either normal or pathological conditions.

Smad signaling pathways regulate pancreatic endocrine development.

Expansion of the pancreatic endocrine cell population occurs during both embryonic development and during post-natal pancreatic growth and regeneration. Mechanisms of the expansion of endocrine cells during embryonic development are not completely understood, and no clear mechanistic link has been established between growth of the embryonic endocrine pancreas and the islet cell replication that occurs in an adult animal. We found that transforming growth factor-beta (TGF-ß) superfamily signaling, which has been implicated in many developmental processes, plays a key role in regulating pancreatic endocrine maturation and development. Specifically, the intracellular mediators of TGF-ß signaling, smad2 and smad3, along with their inhibitor smad7, appear to mediate this process. Smad2, smad3 and smad7 were all broadly expressed throughout the early embryonic pancreatic epithelium. However, during later stages of development, smad2 and smad3 became strongly localized to the nuclei of the endocrine positive cells, whereas the inhibitory smad7 became absent in the endocrine component. Genetic inactivation of smad2 and smad3 led to a significant expansion of the embryonic endocrine compartment, whereas genetic inactivation of smad7 led to a significant decrease in the endocrine compartment. In vitro antisense studies further corroborated these results and supported the possibility that interplay between the inhibitory smad7 and the intracellular mediators smad2/3 is a control point for pancreatic endocrine development. These results should provide a better understanding of the key control mechanisms for ß-cell development.

A simplified purification method for AAV variant by polyethylene glycol aqueous two-phase partitioning.

Adeno-Associated Virus (AAV) has been widely used for in vivo study and preclinical therapy due to its ability to mediate long-term transgene expression, its lack of pathogenicity and low immunogenicity. It has been found that AAV has more than ten serotypes, which each transfect certain types of cells in the viral infected organ. Current methods for purification of different AAV serotypes utilize CsCl or Iodixanol ultrahigh speed density gradient centrifugation, which is expensive and time consuming. We recently developed a simplified method, PEG/(NH(4))(2)SO(4) aqueous two phase partitioning, for purification of AAV serotype 2 and 8. The method does not require ultrahigh speed gradient centrifugation or chromatography. Here we further explore the simplified method for purification of other serotypes of AAV, serotype 6 and 9. This simplified method not only can be used to purify serotype 2 and 8, but also serotype 6 and 9, indicating that a variety of AAV serotypes can be purified by this method.

TGFß receptor signaling is essential for inflammation-induced but not ß-cell workload-induced ß-cell proliferation.

Protection and restoration of a functional ß-cell mass are fundamental strategies for prevention and treatment of diabetes. Consequently, knowledge of signals that determine the functional ß-cell mass is of immense clinical relevance. Transforming growth factor ß (TGFß) superfamily signaling pathways play a critical role in development and tissue specification. Nevertheless, the role of these pathways in adult ß-cell homeostasis is not well defined. Here, we ablated TGFß receptor I and II genes in mice undergoing two surgical ß-cell replication models (partial pancreatectomy or partial duct ligation), representing two triggers for ß-cell proliferation, increased ß-cell workload and local inflammation, respectively. Our data suggest that TGFß receptor signaling is necessary for baseline ß-cell proliferation. By either provision of excess glucose or treatment with exogenous insulin, we further demonstrated that inflammation and increased ß-cell workload are both stimulants for ß-cell proliferation but are TGFß receptor signaling dependent and independent, respectively. Collectively, by using a pancreas-specific TGFß receptor-deleted mouse model, we have identified two distinct pathways that regulate adult ß-cell proliferation. Our study thus provides important information for understanding ß-cell proliferation during normal growth and in pancreatic diseases.

Reduced levels of XPA, ERCC1 and XPF DNA repair proteins in testis tumor cell lines.

Over 80% of patients with advanced metastatic testis tumors can be cured using cisplatin-based combination chemotherapy. This is unusual as metastatic cancer in adults is usually incurable. Cell lines derived from testis tumors retain sensitivity to cisplatin in vitro. We previously investigated 2 testis tumor cell lines with a low capacity to remove cisplatin-induced DNA damage and found that they had low levels of the DNA nucleotide excision repair proteins XPA, ERCC1 and XPF. To determine whether low levels of XPA, ERCC1 and XPF proteins are characteristic of testis tumor cell lines, we investigated 35 cell lines derived from cancers to determine whether groups of cell lines from diverse tissue origins differ from one another in constitutive levels of these NER proteins. Quantitative immunoblotting was used to compare groups of cell lines representing prostate, bladder, breast, lung, cervical, ovarian and testis cancers. Only the 6 testis tumor cell lines showed significantly lower mean levels of XPA (p = 0.001), XPF (p = 0.001) and ERCC1 (p = 0.004) proteins from the other groups. Our results encourage further investigation of the possibility that low levels of these nucleotide excision repair proteins could be related to the favorable response of testis tumors to cisplatin-based chemotherapy.