Arthur V. Sampaio

Roles of IP3R and RyR Ca2+ Channels in Endoplasmic Reticulum Stress and β-Cell Death

OBJECTIVE—Endoplasmic reticulum (ER) stress has been implicated in the pathogenesis of diabetes, but the roles of specific ER Ca2+ release channels in the ER stress–associated apoptosis pathway remain unknown. Here, we examined the effects of stimulating or inhibiting the ER-resident inositol trisphosphate receptors (IP3Rs) and the ryanodine receptors (RyRs) on the induction of β-cell ER stress and apoptosis. RESEARCH DESIGN AND METHODS—Kinetics of β-cell death were tracked by imaging propidium iodide incorporation and caspase-3 activity in real time. ER stress and apoptosis were assessed by Western blot. Mitochondrial membrane potential was monitored by flow cytometry. Cytosolic Ca2+ was imaged using fura-2, and genetically encoded fluorescence resonance energy transfer (FRET)–based probes were used to measure Ca2+ in ER and mitochondria. RESULTS—Neither RyR nor IP3R inhibition, alone or in combination, caused robust death within 24 h. In contrast, blocking sarco/endoplasmic reticulum ATPase (SERCA) pumps depleted ER Ca2+ and induced marked phosphorylation of PKR-like ER kinase (PERK) and eukaryotic initiation factor-2α (eIF2α), C/EBP homologous protein (CHOP)–associated ER stress, caspase-3 activation, and death. Notably, ER stress following SERCA inhibition was attenuated by blocking IP3Rs and RyRs. Conversely, stimulation of ER Ca2+ release channels accelerated thapsigargin-induced ER depletion and apoptosis. SERCA block also activated caspase-9 and induced perturbations of the mitochondrial membrane potential, resulting eventually in the loss of mitochondrial polarization. CONCLUSIONS—This study demonstrates that the activity of ER Ca2+ channels regulates the susceptibility of β-cells to ER stress resulting from impaired SERCA function. Our results also suggest the involvement of mitochondria in β-cell apoptosis associated with dysfunctional β-cell ER Ca2+ homeostasis and ER stress.

Pannexin 3 is a novel target for Runx2, expressed by osteoblasts and mature growth plate chondrocytes.

Pannexins are a class of chordate channel proteins identified by their homology to insect gap junction proteins. The pannexin family consists of three members, Panx1, Panx2, and Panx3, and the role each of these proteins plays in cellular processes is still under investigation. Previous reports of Panx3 expression indicate enrichment in skeletal tissues, so we have further investigated this distribution by surveying the developing mouse embryo with immunofluorescence. High levels of Panx3 were detected in intramembranous craniofacial flat bones, as well as long bones of the appendicular and axial skeleton. This distribution is the result of expression in both osteoblasts and hypertrophic chondrocytes. Furthermore, the Panx3 promoter contains putative binding sites for transcription factors involved in bone formation, and we show that the sequence between bases −275 and −283 is responsive to Runx2 activation. Taken together, our data suggests that Panx3 may serve an important role in bone development, and is a novel target for Runx2‐dependent signaling.

In vivo micro-CT analysis of bone remodeling in a rat calvarial defect model.

The rodent calvarial defect model is commonly used to investigate bone regeneration and wound healing. This study presents a micro-computed tomography (micro-CT) methodology for measuring the bone mineral content (BMC) in a rat calvarial defect and validates it by estimating its precision error. Two defect models were implemented. A single 6 mm diameter defect was created in 20 rats, which were imaged in vivo for longitudinal experiments. Three 5 mm diameter defects were created in three additional rats, which were repeatedly imaged ex vivo to determine precision. Four control rats and four rats treated with bone morphogenetic protein were imaged at 3, 6, 9 and 12 weeks post-surgery. Scan parameters were 80 kVp, 0.45 mA and 180 mAs. Images were reconstructed with an isotropic resolution of 45 microm. At 6 weeks, the BMC in control animals (4.37 +/- 0.66 mg) was significantly lower (p < 0.05) than that in treated rats (11.29 +/- 1.01 mg). Linear regression between the BMC and bone fractional area, from 20 rats, showed a strong correlation (r(2) = 0.70, p < 0.0001), indicating that the BMC can be used, in place of previous destructive analysis techniques, to characterize bone growth. The high precision (2.5%) of the micro-CT methodology indicates its utility in detecting small BMC changes in animals.

Scleraxis expression is coordinately regulated in a murine model of patellar tendon injury.

This study investigated the expression of Scleraxis in a murine model of patellar tendon injury in which the central third of the patellar tendon was unilaterally injured. The presence of tendon pathology was assessed using dual photon microscopy, conventional histology and microCT. Tendon pathology was also quantified noninvasively over a 12‐week period using high‐frequency ultrasound and laser Doppler flowmetry. Gene expression (Scx, Tnmd, and Col1a1) was determined at defined end‐points (1, 4, 8, and 12 weeks) using qPCR on RNA from individual patellar tendons on injured and uninjured sides. There was significant development of tendon pathology as gauged by ultrasound and laser Doppler over 12 weeks. Injured tendons demonstrated significant histological and microCT evidence of pathological change, and disorganized collagen with reduced density. The expression of Scx and Col1a1 was unchanged at 1 week, significantly upregulated at 4 and 8 weeks, and had returned to baseline by 12 weeks. Tnmd expression was unchanged at 1 week, and significantly increased at 4, 8, and 12 weeks. Patellar tendon injury was associated with marked increases in the expression of Scx, Tnmd, and Col1a1. Our data suggest new roles for Scleraxis in coordinating the response to injury in the pathogenesis of tendon disorders.

EGR1 reactivation by histone deacetylase inhibitors promotes synovial sarcoma cell death through the PTEN tumor suppressor.

Synovial sarcoma is a high-grade soft tissue malignancy, for which current cytotoxic chemotherapies provide limited benefit. Although histone deacetylase (HDAC) inhibitors are known to suppress synovial sarcoma in vitro and in vivo, the exact mechanism is not clear. In this study, we report a central role of the transcription factor, early growth response-1 (EGR1), in the regulation of HDAC inhibitor-induced apoptotic cell death in synovial sarcoma. The SS18-SSX oncoprotein, characteristic of synovial sarcoma, maintains EGR1 expression at low levels, whereas it is significantly increased after HDAC inhibitor treatment. On the contrary, EGR1 knockdown leads to a decrease in HDAC inhibitor-induced apoptosis. Moreover, we find that under these conditions phosphatase and tensin homolog deleted in chromosome 10 (PTEN) is upregulated and this occurs through direct binding of EGR1 to an element upstream of the PTEN promoter. Using a combination of gain- and loss-of-function approaches, we show that EGR1 modulation of PTEN contributes to HDAC inhibitor-induced apoptosis in synovial sarcoma. Finally, restoration of EGR1 or PTEN expression is sufficient to induce synovial sarcoma cell death. Taken together, our findings indicate that SS18-SSX-mediated attenuation of an EGR1–PTEN network regulates synovial sarcoma cell survival, and that HDAC inhibitor-mediated apoptosis operates at least in part through reactivation of this pathway.

Functionally Convergent White Adipogenic Progenitors of Different Lineages Participate in a Diffused System Supporting Tissue Regeneration

Pathologies characterized by lipomatous infiltration of craniofacial structures as well as certain forms of lipodystrophies suggest the existence of a distinct adipogenic program in the cephalic region of mammals. Using lineage tracing, we studied the origin of craniofacial adipocytes that accumulate both in cranial fat depots and during ectopic lipomatous infiltration of craniofacial muscles. We found that unlike their counterparts in limb muscle, a significant percentage of cranial adipocytes is derived from the neural crest (NC). In addition, we identified a population of NC‐derived Lin−/α7−/CD34+/Sca‐1+ fibro/adipogenic progenitors (NC‐FAPs) that resides exclusively in the mesenchyme of cephalic fat and muscle. Comparative analysis of the adipogenic potential, impact on metabolism, and contribution to the regenerative response of NC‐FAPs and mesoderm‐derived FAPs (M‐FAPs) suggests that these cells are functionally indistinguishable. While both NC‐ and M‐FAPs express mesenchymal markers and promyogenic cytokines upon damage‐induced activation, NC‐FAPs additionally express components of the NC developmental program. Furthermore, we show that craniofacial FAP composition changes with age, with young mice containing FAPs that are almost exclusively of NC origin, while NC‐FAPs are progressively replaced by M‐FAPs as mice age. Based on these results, we propose that in the adult, ontogenetically distinct FAPs form a diffused system reminiscent of the endothelium, which can originate from multiple developmental intermediates to seed all anatomical locations. STEM CELLS2012;30:1152–1162

Genetic deletion of Cyp26b1 negatively impacts limb skeletogenesis by inhibiting chondrogenesis

Cyp26b1, a retinoic acid (RA)-metabolising enzyme, is expressed in the developing limb bud, and Cyp26b1−/− mice present with severe limb defects. These malformations might be attributable to an RA-induced patterning defect; however, recent reports suggest that RA is dispensable for limb patterning. In this study, we examined the role of endogenous retinoid signalling in skeletogenesis using Cyp26b1−/− mice and transgenic mice in which Cyp26b1 is conditionally deleted under control of the Prrx1 promoter beginning at ~E9.5 (Prrx1Cre+/Cyp26b1fl/fl). We found that the limb phenotype in Prrx1Cre+/Cyp26b1fl/fl mice was less severe than that observed in Cyp26b1−/− animals and that a change in retinoid signalling contributed to the difference in phenotypes. We systematically examined the role of endogenous RA signalling in chondrogenesis and found that Cyp26b1−/− cells and limb mesenchymal cells treated with a CYP inhibitor, are maintained in a pre-chondrogenic state, exhibit reduced chondroblast differentiation and have modestly accelerated chondrocyte hypertrophy. Furthermore, Cyp26b1−/− mesenchyme exhibited an increase in expression of genes in a closely related tendogenic lineage, indicating that retinoid signals in the limb interfere with differentiation and maintain progenitor status. Together, these findings support an important function for RA in regulating the behaviour of mesenchymal progenitors, and their subsequent differentiation and maturation.

Noncanonical Activation of Akt/Protein Kinase B in β-Cells by the Incretin Hormone Glucose-dependent Insulinotropic Polypeptide

Therapeutics based on the actions of the incretin hormones, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), have recently been introduced for the treatment of type 2 diabetes mellitus. The serine/threonine kinase Akt is a major mediator of incretin action on the pancreatic islet, increasing β-cell mass and function and promoting β-cell survival. The mechanisms underlying incretin activation of Akt are thought to involve an essential phosphoinositide 3-kinase-mediated phosphorylation of threonine 308, similar to the prototypical Akt activator, insulin-like growth factor-I (IGF-I). In this study, using activity assays on immunoprecipitated Akt, we discovered that GIP and GLP-1 were capable of stimulating Akt in the INS-1 β-cell line and isolated mouse islets via a mechanism that did not require phosphoinositide 3-kinase or phosphorylation of Thr308 and Ser473, and this pathway involved the production of cAMP. Furthermore, we found that GIP stimulated anti-apoptotic signaling via this alternate mode of Akt activation. We conclude that incretins can activate Akt via a novel noncanonical mechanism that may provide an alternative therapeutic target for the treatment of type 2 diabetes mellitus and have broader implications for Akt physiology in human health and disease.

Non-canonical activation of Akt/PKB in β-cells by the incretin hormone glucose-dependent insulinotropic polypeptide (GIP)

Therapeutics based on the actions of the incretin hormones, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), have recently been introduced for the treatment of type 2 diabetes mellitus. The serine/threonine kinase Akt is a major mediator of incretin action on the pancreatic islet, increasing β-cell mass and function and promoting β-cell survival. The mechanisms underlying incretin activation of Akt are thought to involve an essential phosphoinositide 3-kinase-mediated phosphorylation of threonine 308, similar to the prototypical Akt activator, insulin-like growth factor-I (IGF-I). In this study, using activity assays on immunoprecipitated Akt, we discovered that GIP and GLP-1 were capable of stimulating Akt in the INS-1 β-cell line and isolated mouse islets via a mechanism that did not require phosphoinositide 3-kinase or phosphorylation of Thr308 and Ser473, and this pathway involved the production of cAMP. Furthermore, we found that GIP stimulated anti-apoptotic signaling via this alternate mode of Akt activation. We conclude that incretins can activate Akt via a novel noncanonical mechanism that may provide an alternative therapeutic target for the treatment of type 2 diabetes mellitus and have broader implications for Akt physiology in human health and disease.

Sodium cromolyn reduces expression of CTGF, ADAMTS1, and TIMP3 and modulates post-injury patellar tendon morphology

The purpose of this study was to determine whether administration of a mast cell inhibitor (sodium cromolyn, SC) would influence tendon repair and extracellular matrix gene expression following acute injury. CD1 mouse patellar tendons were unilaterally injured and mast cell prevalence was determined. The effect of SC injection on tendon hypercellularity, cross‐sectional area, collagen organization, and expression of extracellular matrix‐related genes was examined. Mast cell prevalence was markedly increased in injured patellar tendons (p = 0.009), especially at 8 weeks post‐injury (p = 0.025). SC injection increased collagen organization compared to uninjected animals at 4 weeks and attenuated the development of tendon hypercellularity and tendon thickening post‐injury. Expression of CTGF, ADAMTS1, and TIMP3 in injured tendon was reduced in the SC group. SC injections moderated the structural alterations of healing tendon in association with downregulation of several genes associated with tendon fibrosis. This work corroborates previous findings pointing to a role of mast cells in tendon repair.