Richard Blouin

Adiponectin action from head to toe.

Adiponectin, the most abundant protein secreted by white adipose tissue, is known for its involvement in obesity-related disorders such as insulin resistance, type 2 diabetes mellitus and atherosclerosis. Moreover, modulation of the circulating adiponectin concentration is observed in pathologies that are more or less obesity-related, such as cancer and rheumatoid arthritis. The wide distribution of adiponectin receptors in various organs and tissues suggests that adiponectin has pleiotropic effects on numerous physiological processes. Besides its well-known insulin-sensitizing, anti-inflammatory and antiatherosclerotic properties, accumulating evidence suggests that adiponectin may also have anticancer properties and be cardioprotective. A beneficial effect of adiponectin on female reproductive function was also suggested. Since adiponectin has numerous beneficial biological functions, its use as a therapeutic agent has been suggested. However, the use of adiponectin or its receptors as therapeutic targets is complicated by the presence of different adiponectin oligomeric isoforms and production sites, by multiple receptors with differing affinities for adiponectin isoforms, and by cell-type-specific effects in different tissues. In this review, we discuss the known and potential roles of adiponectin in various tissues and pathologies. The therapeutic promise of administration of adiponectin and the use of its circulating levels as a diagnostic biomarker are further discussed based on the latest experimental studies.

Functional genomic screening identifies dual leucine zipper kinase as a key mediator of retinal ganglion cell death

Glaucoma, a major cause of blindness worldwide, is a neurodegenerative optic neuropathy in which vision loss is caused by loss of retinal ganglion cells (RGCs). To better define the pathways mediating RGC death and identify targets for the development of neuroprotective drugs, we developed a high-throughput RNA interference screen with primary RGCs and used it to screen the full mouse kinome. The screen identified dual leucine zipper kinase (DLK) as a key neuroprotective target in RGCs. In cultured RGCs, DLK signaling is both necessary and sufficient for cell death. DLK undergoes robust posttranscriptional up-regulation in response to axonal injury in vitro and in vivo. Using a conditional knockout approach, we confirmed that DLK is required for RGC JNK activation and cell death in a rodent model of optic neuropathy. In addition, tozasertib, a small molecule protein kinase inhibitor with activity against DLK, protects RGCs from cell death in rodent glaucoma and traumatic optic neuropathy models. Together, our results establish a previously undescribed drug/drug target combination in glaucoma, identify an early marker of RGC injury, and provide a starting point for the development of more specific neuroprotective DLK inhibitors for the treatment of glaucoma, nonglaucomatous forms of optic neuropathy, and perhaps other CNS neurodegenerations.

Sanguinarine induces apoptosis of human osteosarcoma cells through the extrinsic and intrinsic pathways.

The quaternary benzo[c]phenanthridine alkaloid sanguinarine inhibits the proliferation of cancerous cells from different origins, including lung, breast, pancreatic and colon, but nothing is known of its effects on osteosarcoma, a primary malignant bone tumour. We have found that sanguinarine alters the morphology and reduces the viability of MG-63 and SaOS-2 human osteosarcoma cell lines in concentration- and time-dependent manner. Incubation with 1 micromol/L sanguinarine for 4 and 24h killed more efficiently MG-63 cells than SaOS-2 cells, while incubation with 5 micromol/L sanguinarine killed almost 100% of both cell populations within 24h. This treatment also changed the mitochondrial membrane potential in both MG-63 and SaOS-2 cells within 1h, caused chromatin condensation and the formation of apoptotic bodies. It activated multicaspases, and increased the activities of caspase-8 and caspase-9 in both MG-63 and SaOS-2 cells. These data highlight sanguinarine as a novel potential agent for bone cancer therapy.

The Mixed Lineage Kinase DLK Is Oligomerized by Tissue Transglutaminase during Apoptosis

Current evidence suggests that the mixed lineage kinase family member dual leucine zipper-bearing kinase (DLK) might play a significant role in the regulation of cell growth and differentiation, particularly during the process of tissue remodeling. To further explore this working model, we have investigated the regulation of host and recombinant DLK in NIH3T3 and COS-1 cells undergoing apoptosis. Using calphostin C, a potent and selective inhibitor of protein kinase C and a recognized apoptosis inducer for various cell types, we demonstrate, by immunoblot analysis, that DLK protein levels are rapidly and dramatically down-regulated during the early phases of apoptosis. Down-regulation in calphostin C-treated cells was also accompanied by the appearance of SDS- and mercaptoethanol-resistant high molecular weight DLK immunoreactive oligomers. Experiments aimed at elucidating the mechanism(s) underlying DLK oligomerization revealed that the tissue transglutaminase (tTG) inhibitor monodansylcadaverine antagonized the effects of calphostin C almost completely, thereby suggesting the involvement of a tTG-catalyzed reaction as the root cause of DLK down-regulation and accumulation as high molecular weight species. In support of this notion, we also show that DLK can serve as a substrate for tTG-dependent cross-linking in vitro and that this covalent post-translational modification leads to the functional inactivation of DLK. Taken together, these observations suggest that transglutamination and oligomerization may constitute a relevant physiological mechanism for the regulation of DLK activity.

Down-regulation of the Mixed-lineage Dual Leucine Zipper-bearing Kinase by Heat Shock Protein 70 and Its Co-chaperone CHIP

Dual leucine zipper-bearing kinase (DLK) is a mixed-lineage kinase family member that acts as an upstream activator of the c-Jun N-terminal kinases. As opposed to other components of this pathway, very little is currently known regarding the mechanisms by which DLK is regulated in mammalian cells. Here we identify the stress-inducible heat shock protein 70 (Hsp70) as a negative regulator of DLK expression and activity. Support for this notion derives from data showing that Hsp70 induces the proteasomal degradation of DLK when both proteins are co-expressed in COS-7 cells. Hsp70-mediated degradation occurs with expression of wild-type DLK, which functions as a constitutively activated protein in these cells but not kinase-defective DLK. Interestingly, the Hsp70 co-chaperone CHIP, an E3 ubiquitin ligase, seems to be indispensable for this process since Hsp70 failed to induce DLK degradation in COS-7 cells expressing a CHIP mutant unable to catalyze ubiquitination or in immortalized fibroblasts derived from CHIP knock-out mice. Consistent with these data, we have found that endogenous DLK becomes sensitive to CHIP-dependent proteasomal degradation when it is activated by okadaic acid and that down-regulation of Hsp70 levels with an Hsp70 antisense attenuates this sensitivity. Therefore, our studies suggest that Hsp70 contributes to the regulation of activated DLK by promoting its CHIP-dependent proteasomal degradation.

The mitogen-activated protein kinase kinase kinase dual leucine zipper-bearing kinase (DLK) acts as a key regulator of keratinocyte terminal differentiation

In the skin, epithelial cells undergo a terminal differentiation program leading to the formation of the stratum corneum. Although it is expected that the last phases of this process must be tightly regulated since it results in cell death, the signaling pathways involved in this induction remain ill defined. We now report that a single kinase, the mitogen-activated protein kinase kinase kinase dual leucine zipper-bearing kinase (DLK), acts in the epidermis to promote the terminal differentiation of human keratinocytes. In support of this notion, we showed that DLK expression was restricted to the granular layer in situ. In addition, cultured keratinocytes infected with a recombinant adenovirus expressing DLK exhibited morphological and biochemical changes, including a suprabasal localization, altered cell shape, compacted cytoplasm, DNA fragmentation, and the up-regulation of filaggrin, that are reminiscent of a terminally differentiated phenotype. Moreover the expression of wild-type DLK in keratinocytes stimulated transglutaminase activity and the consequent formation of the cornified cell envelope, while a kinase-inactive variant of DLK did not. Together these results identify DLK as a signaling molecule implicated in the regulation of keratinocyte terminal differentiation and cornification.

The Small Heat-Shock Protein Hsp27 Undergoes ERK-Dependent Phosphorylation and Redistribution to the Cytoskeleton in Response to Dual Leucine Zipper-Bearing Kinase Expression

Hsp27, a small heat-shock protein, has important roles in many cellular processes, including cytoskeleton dynamics, cell differentiation, and apoptosis. Its expression in normal epidermis correlates with differentiation; however, little is known about the regulatory mechanisms involved. In this study, we report that Hsp27 undergoes upregulation, phosphorylation, and redistribution to the cytoskeleton during the late phase of epidermal keratinocyte differentiation. Our results also show that the expression of the dual leucine zipper-bearing kinase (DLK), an upstream activator of the MAP kinase pathways, is sufficient by itself to induce Hsp27 phosphorylation, cell periphery localization, and redistribution to the insoluble protein fraction (cytoskeleton) in poorly differentiated keratinocytes. This redistribution correlates with the insolubilization of cornified envelope-associated proteins such as involucrin. Interestingly, the effects of DLK on Hsp27 were blocked by PD98059, a selective inhibitor of the extracellular signal-regulated protein kinase (ERK) pathway. Moreover, downregulation of Hsp27 by small interfering RNA in epithelial cells expressing DLK was accompanied by attenuated expression of involucrin in the cytoskeleton. Thus, these observations suggest that the DLK-ERK signaling pathway may act as a regulator of the interaction that occurs between Hsp27 and the cytoskeleton during the formation of the cornified cell envelope, a process conferring to the skin its crucial barrier function.

Isolation of Differentially Expressed Genes in Conceptuses and Endometrial Tissue of Sows in Early Gestation

Abstract The implantation period is a critical time for embryonic survival in pigs. During this period, numerous growth factors are secreted by the conceptuses and the uterine endometrium in order to establish pregnancy and to provide a proper environment for embryonic development. It is well known that the Chinese Meishan sows have a larger litter size when compared with occidental sows mainly because of a superior embryonic survival rate. As a further step toward understanding the mechanisms involved in embryonic survival, we used a suppression subtractive hybridization technique to identify genes that were differentially expressed in Meishan-Landrace conceptuses and endometrial tissue at Day 15 of gestation when compared with conventional Landrace sows. Of the 1000 subtractive clones isolated from each library, 137 endometrial and 166 conceptus-enriched cDNAs were single-pass sequenced and examined by BLAST analysis for identification. Sixty-two percent of the clones found in the endometrial library and 78% of the clones found in the conceptus library showed homology with known genes. Among these genes, the 20 most relevant to embryonic survival based on the available literature were validated through real-time reverse-transcriptase polymerase chain reaction (RT-PCR) analysis. Our results show that suppression subtractive hybridization is a powerful method applicable in identifying putative candidate genes that might be used for selection of high litter-size breeds.

Overexpression of the reg Gene In Non-obese Diabetic Mouse Pancreas During Active Diabetogenesis Is Restricted to Exocrine Tissue

We demonstrated pancreatic reg gene overexpression in non-obese diabetic (NOD) mice during active diabetogenesis. The aim of this study was to determine in which part of the pancreas (endocrine and/or exocrine) the gene(s) and the protein(s) were expressed and if their localization changed with progression of the disease. In situ hybridization analysis and immunocytochemical studies were carried out on pancreas of female and male NOD mice. Both develop insulitis but diabetes develops only in females and in males only when treated by cyclophosphamide. Our results show that whatever the age, sex, and presence of insulitis and/or diabetes, the expression of reg mRNAs and of the corresponding protein(s) was restricted to exocrine tissue. Moreover, reg remains localized in acinar cells in the two opposite situations of (a) cyclophosphamide-treated males in a prediabetic stage presenting a high level of both insulin and reg mRNAs, and (b) the overtly diabetic females with no insulin but a high level of reg mRNA. These findings suggest that overexpression of the reg gene(s) might represent a defense of the acinar cell against pancreatic aggression.

Transglutaminase 2 facilitates or ameliorates HIF signaling and ischemic cell death depending on its conformation and localization

Transglutaminase 2 (TG2) is a widely expressed and multifunctional protein that modulates cell death/survival processes. We have previously shown that TG2 binds to hypoxia inducible factor 1β (HIF1β) and decreases the upregulation of HIF responsive genes; however, the relationship between these observations was not investigated. In this study, we investigated whether endogenous TG2 is sufficient to suppress HIF activity and whether the interaction between TG2 and HIF1β is required for this suppression. shRNA-mediated silencing of TG2 significantly enhanced HIF activation in response to hypoxia. In addition, nuclear localization of TG2 is required for its suppressive effect on HIF activity, with TG2 being recruited to HIF responsive promoters in hypoxic conditions. These observations suggest that TG2 directly regulates hypoxic transcriptional machinery; however, its interaction with HIF1β was not required for this regulation. We also examined whether TG2s effect on cell death/survival processes in ischemia is due to its effects on HIF signaling. Our results indicate that TG2 mediated HIF suppression can be separated from TG2s effect on cell survival in hypoxic/hypoglycemic conditions. Lastly, here we show that nuclear TG2 in the closed conformation and non-nuclear TG2 in the open conformation have opposing effects on hypoxic/hypoglycemic cell death, which could explain previous controversial results. Overall, our results further clarify the role of TG2 in mediating the cellular response to ischemia and suggest that manipulating the conformation of TG2 might be of pharmacological interest as a therapeutic strategy for the treatment of ischemia-related pathologies.