The stromal cell–derived factor-1α/CXCR4 ligand–receptor axis is critical for progenitor survival and migration in the pancreas

The SDF-1α/CXCR4 ligand/chemokine receptor pair is required for appropriate patterning during ontogeny and stimulates the growth and differentiation of critical cell types. Here, we demonstrate SDF-1α and CXCR4 expression in fetal pancreas. We have found that SDF-1α and its receptor CXCR4 are expressed in islets, also CXCR4 is expressed in and around the proliferating duct epithelium of the regenerating pancreas of the interferon (IFN) γ–nonobese diabetic mouse. We show that SDF-1α stimulates the phosphorylation of Akt, mitogen-activated protein kinase, and Src in pancreatic duct cells. Furthermore, migration assays indicate a stimulatory effect of SDF-1α on ductal cell migration. Importantly, blocking the SDF-1α/CXCR4 axis in IFNγ-nonobese diabetic mice resulted in diminished proliferation and increased apoptosis in the pancreatic ductal cells. Together, these data indicate that the SDF-1α–CXCR4 ligand receptor axis is an obligatory component in the maintenance of duct cell survival, proliferation, and migration during pancreatic regeneration

The stromal cell-derived factor-1alpha/CXCR4 ligand-receptor axis is critical for progenitor survival and migration in the pancreas.

The SDF-1alpha/CXCR4 ligand/chemokine receptor pair is required for appropriate patterning during ontogeny and stimulates the growth and differentiation of critical cell types. Here, we demonstrate SDF-1alpha and CXCR4 expression in fetal pancreas. We have found that SDF-1alpha and its receptor CXCR4 are expressed in islets, also CXCR4 is expressed in and around the proliferating duct epithelium of the regenerating pancreas of the interferon (IFN) gamma-nonobese diabetic mouse. We show that SDF-1alpha stimulates the phosphorylation of Akt, mitogen-activated protein kinase, and Src in pancreatic duct cells. Furthermore, migration assays indicate a stimulatory effect of SDF-1alpha on ductal cell migration. Importantly, blocking the SDF-1alpha/CXCR4 axis in IFNgamma-nonobese diabetic mice resulted in diminished proliferation and increased apoptosis in the pancreatic ductal cells. Together, these data indicate that the SDF-1alpha-CXCR4 ligand receptor axis is an obligatory component in the maintenance of duct cell survival, proliferation, and migration during pancreatic regeneration

The SDF-1α/CXCR4 Axis is Required for Proliferation and Maturation of Human Fetal Pancreatic Endocrine Progenitor Cells

The chemokine receptor CXCR4 and ligand SDF-1α are expressed in fetal and adult mouse islets. Neutralization of CXCR4 has previously been shown to diminish ductal cell proliferation and increase apoptosis in the IFNγ transgenic mouse model in which the adult mouse pancreas displays islet regeneration. Here, we demonstrate that CXCR4 and SDF-1α are expressed in the human fetal pancreas and that during early gestation, CXCR4 colocalizes with neurogenin 3 (ngn3), a key transcription factor for endocrine specification in the pancreas. Treatment of islet like clusters (ICCs) derived from human fetal pancreas with SDF-1α resulted in increased proliferation of epithelial cells in ICCs without a concomitant increase in total insulin expression. Exposure of ICCs in vitro to AMD3100, a pharmacological inhibitor of CXCR4, did not alter expression of endocrine hormones insulin and glucagon, or the pancreatic endocrine transcription factors PDX1, Nkx6.1, Ngn3 and PAX4. However, a strong inhibition of β cell genesis was observed when in vitro AMD3100 treatment of ICCs was followed by two weeks of in vivo treatment with AMD3100 after ICC transplantation into mice. Analysis of the grafts for human C-peptide found that inhibition of CXCR4 activity profoundly inhibits islet development. Subsequently, a model pancreatic epithelial cell system (CFPAC-1) was employed to study the signals that regulate proliferation and apoptosis by the SDF-1α/CXCR4 axis. From a selected panel of inhibitors tested, both the PI 3-kinase and MAPK pathways were identified as critical regulators of CFPAC-1 proliferation. SDF-1α stimulated Akt phosphorylation, but failed to increase phosphorylation of Erk above the high basal levels observed. Taken together, these results indicate that SDF-1α/CXCR4 axis plays a critical regulatory role in the genesis of human islets

Role of p38 Mitogen-Activated Protein Kinase in Middle Ear Mucosa Hyperplasia during Bacterial Otitis Media

Hyperplasia of the middle ear mucosa contributes to the sequelae of acute otitis media. Understanding the signal transduction pathways that mediate hyperplasia could lead to the development of new therapeutic interventions for this disease and its sequelae. Endotoxin derived from bacteria involved in middle ear infection can contribute to the hyperplastic response. The p38 mitogen-activated protein kinase (MAPK) is known to be activated by endotoxin as well as cytokines and other inflammatory mediators that have been documented in otitis media. We assessed the activation of p38 in the middle ear mucosa of an in vivo rat bacterial otitis media model. Strong activity of p38 was observed 1 to 6 h after bacterial inoculation. Activity continued at a lower level for at least 7 days. The effects of p38 activation were assessed using an in vitro model of rat middle ear mucosal hyperplasia in which mucosal growth is stimulated by nontypeable Haemophilus influenzae during acute otitis media. Hyperplastic mucosal explants treated with the p38α and p38β inhibitor SB203580 demonstrated significant inhibition of otitis media-stimulated mucosal growth. The results of this study suggest that intracellular signaling via p38 MAPK influences the hyperplastic response of the middle ear mucosa during bacterial otitis media

Limited Capacity of Human Adult Islets Expanded In Vitro to Redifferentiate Into Insulin-Producing β-cells

Limited organ availability is an obstacle to the widespread use of islet transplantation in type 1 diabetic patients. To address this problem, many studies have explored methods for expanding functional human islets in vitro for diabetes cell therapy. We previously showed that islet cells replicate after monolayer formation under the influence of hepatocyte growth factor and selected extracellular matrices. However, under these conditions, senescence and loss of insulin expression occur after >15 doublings. In contrast, other groups have reported that islet cells expanded in monolayers for months progressed through a reversible epithelial-to-mesenchymal transition, and that on removal of serum from the cultures, islet-like structures producing insulin were formed (1). The aim of the current study was to compare the two methods for islet expansion using immunostaining, real-time quantitative PCR, and microarrays at the following time points: on arrival, after monolayer expansion, and after 1 week in serum-free media. At this time, cell aliquots were grafted into nude mice to study in vivo function. The two methods showed similar results in islet cell expansion. Attempts at cell differentiation after expansion by both methods failed to consistently recover a β-cell phenotype. Redifferentiation of β-cells after expansion is still a challenge in need of a solution.Fil: Kayali, Ayse G. University of California at San Diego; Estados UnidosFil: Flores, Luis Emilio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. University of California at San Diego; Estados UnidosFil: Lopez, Ana D.. University of California at San Diego; Estados UnidosFil: Kutlu, Burak. Institute for Systems Biology; Estados UnidosFil: Baetge, Emmanuel. Novocell; Estados UnidosFil: Kitamura, Ryuichi. University of California at San Diego; Estados UnidosFil: Hao, Ergeng. University of California at San Diego; Estados UnidosFil: Beattie, Gillian M.. University of California at San Diego; Estados UnidosFil: Hayek, Alberto. University of California at San Diego; Estados Unido

Expression of insulin and glucagon in grafts from control and AMD3100 treated ICCs transplanted into nu/nu mice.

<p>Double immunofluorescent staining of insulin (green) and glucagon (red) in ICCs transplanted under the kidney capsule of nu/nu athymic mice treated with saline (A, B) or AMD3100 every two days during two weeks following transplantation. Insulin and glucagon expression is extensive in the control ICCs. C. Insulin or glucagon was not detectable in the grafts from the mice treated with AMD3100. Note the presence of the round beads that were inserted to identify the transplant site.</p

SDF-1 stimulates proliferation of epithelial cells in ICCs from early gestational human fetal pancreas.

<p>ICCs isolated from fetal pancreas of 12 to 14 weeks were grown in suspension culture, treated with 10% human serum alone, with HGF (10 ng/ml) or with SDF-1α (100 ng/ml) for six days. ICCs were fixed and analyzed by immunofluorescence staining. PanCytokeratin (green) is an epithelial marker. Ki67 (red) is used as a marker of proliferation.</p

SDF-1α stimulates Akt but not MAPK phosphorylation in CFPAC-1 cells and fetal ICCs.

<p>Following 48 hr serum starvation, CFPAC-1 cells were stimulated with 100 ng/ml or 300 ng/ml human recombinant SDF-1α for 10 min at 37°C. Whole cell lystes were analyzed by western blot, using antibodies raised against dually phosphorylated phospho-MAPK(ERK1/ERK2)(A) or phospho-Akt (Ser473)(B). Following overnight serum starvation, ICCs from human fetal pancreas of 15 weeks gestation were stimulated with 100 ng/ml or 300 ng/ml SDF-1α for 10 min at 37°C. Whole cell lystes were analyzed by western blot, using an antibody raised against phospho-MAPK(C) or phospho-Akt (Ser473) (D). All blots were stripped and reblotted with antibodies to total Akt, Erk, and Hsp90 sequentially to confirm equal loading.</p

The effect of the inhibitors of PI 3-kinase, MAPK, PLC, and PKA on SDF-1α stimulated proliferation in CFPAC-1 cells.

<p>CFPAC-1 cells were seeded in 12 well plates and grown to 50–70% confluence. Following 24 hr serum starvation, cells were pre-treated with LY294002 (30 µM), U0126 (30 µM), Edelfosine (10 µM), or H89 (10 µM) for 30 minutes and stimulated with SDF-1α for 16 hrs. BrdU was added 4 hrs before fixation. BrdU incorporation was determined as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038721#s4" target="_blank">Materials and Methods</a>. (*, P<0.05 SDF-1 versus basal); (**, p<0.02 SDF-1/Edelfosine versus SDF-1 alone); (***, p<0.0001 SDF-1/LY294002 and SDF-1/U0216 versus SDF-1 alone) by Student’s t-test. N.S.  =  not significant by Student’s t-test.</p