Alex Daviau

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.

Nanoparticle-mediated growth factor delivery systems: A new way to treat Alzheimer's disease

The number of people diagnosed with Alzheimers disease (AD) is increasing steadily as the world population ages, thus creating a huge socio-economic burden. Current treatments have only transient effects and concentrate on a single aspect of AD. There is much evidence suggesting that growth factors (GFs) have a great therapeutic potential and can play on all AD hallmarks. Because GFs are prone to denaturation and clearance, a delivery system is required to ensure protection and a sustainable delivery. This review provides information about the latest advances in the development of GF delivery systems (GFDS) targeting the brain in terms of in vitro and in vivo effects in the context of AD and discusses new strategies designed to increase the availability and the specificity of GFs to the brain. This paper also discusses, on a mechanistic level, the different delivery hurdles encountered by the carrier or the GF itself from its injection site up to the brain tissue. The major mass transport phenomena influencing the delivery systems targeting the brain are addressed and insights are given about how mechanistic mathematical frameworks can be developed to use and optimize them.

Effect of BMP-2 and/or BMP-9 on preosteoblasts attached to polycaprolactone functionalized by adhesive peptides derived from bone sialoprotein

Biomaterials functionalized by adhesive peptides improve the cell-substratum interaction. However, their influence on the response of cells to growth factors is still poorly understood. We have shown that bone morphogenetic protein (BMP) 2 activates the Smad pathway only in murine MC3T3-E1 preosteoblasts attached to polycaprolactone (PCL) film functionalized by RGD peptides derived from bone sialoprotein (pRGD). We have now analysed the way recombinant human BMP-2 and/or BMP-9 (0.38 nM) influence the signal transduction and differentiation of MC3T3-E1 preosteoblasts attached to PCL-pRGD. While kinetics of MAPK activation were similar in cells treated by BMP-2 and BMP-9, different kinetics of Smad activation and β-catenin stabilization were observed. BMP-2 induced Smad1/5/8 phosphorylation within 0.5 and BMP-9 within 4 h, while the β-catenin was lower at 2 h only in cells treated with BMP-9. However, both BMPs induced the translocation of phosphorylated Smad1/5/8 to the nucleus at 4 h and increased Dlx5, osterix and osteocalcin transcripts as well as alkaline phosphatase activity at 72 h. A BMP-2/BMP-9 combination that maintained the β-catenin amount constant but reduced that of phosphorylated Smad within 4 h had quite similar effect than BMP-2 alone. It is therefore important to determine how biomimetic materials influence the response of cells to BMPs.

The mixed-lineage kinase DLK is a key regulator of 3T3-L1 adipocyte differentiation.

Background The mixed-lineage kinase (MLK) family member DLK has been proposed to serve as a regulator of differentiation in various cell types; however, its role in adipogenesis has not been investigated. In this study, we used the 3T3-L1 preadipocyte cell line as a model to examine the function of DLK in adipocyte differentiation. Methods and Findings Immunoblot analyses and kinase assays performed on 3T3-L1 cells showed that the expression and activity of DLK substantially increase as differentiation occurs. Interestingly, DLK appears crucial for differentiation since its depletion by RNA interference impairs lipid accumulation as well as expression of the master regulators of adipogenesis C/EBPα and PPARγ2 at both the mRNA and protein levels. In contrast, neither the expression nor the DNA binding activity of C/EBPβ, an activator for C/EBPα and PPARγ, is affected by DLK loss. Conclusions Taken together, these results suggest that DLK is important for expression of mature adipocyte markers and that its action most likely takes place via regulation of C/EBPβ transcriptional activity and/or initiation of C/EBPα and PPARγ2 gene transcription.

Modulation of MAPK signalling by immobilized adhesive peptides: Effect on stem cell response to BMP-9-derived peptides.

UNLABELLED Biomimetic materials were developed to regulate stem cell behaviour. We have analyzed the influence of polycaprolactone (PCL) films, functionalized with adhesive peptides derived from fibronectin (pFibro) or bone sialoprotein (pBSP), on the response of murine multipotent C3H10T1/2 cells to bone morphogenetic protein-9 (BMP-9) and its derived peptides (pBMP-9 and SpBMP-9). PCL-pFibro promoted better cell cytoskeleton organization and faster focal adhesion kinase activation than did PCL-pBSP. PCL-pFibro also promoted MAPK signalling to improve the cell response to BMP-9 by inactivating ERK1/2 and stimulating p38 and JNK. BMP-9, pBMP-9 and SpBMP-9 induced greater phosphorylation of Smad1/5/8 in cells attached to PCL-pFibro than in cells on PCL-pBSP. These phosphorylated Smad1/5/8 were translocated to the nucleus. BMP-9 and its derived peptides restored the phosphorylation of JNK in cells on PCL-pBSP, but it remained less phosphorylated than in cells on PCL-pFibro stimulated with pBMP-9 and SpBMP-9. Cells attached to PCL-pFibro contained more Runx2, essential for stem cell commitment to become osteoblasts, than did cells on PCL-pBSP when incubated with BMP-9 and its derived peptides. Runx2 was no longer detected when the cells were pre-treated with JNK inhibitor. Therefore pFibro plus BMP-9 and its derived peptides may be a promising strategy to develop biomimetic materials. STATEMENT OF SIGNIFICANCE Biomaterials functionalized with adhesive peptides to favour bone repair have generated a great interest over the past decade. However, the effect of these materials on the ability of cells to respond to growth factors remains poorly known. One major growth factor subfamily involved in bone formation is the bone morphogenetic protein (BMP). However, these BMPs are expensive. We therefore developed less costly derived molecules. We showed how adhesive peptides derived from bone matrix proteins grafted onto polymer films affect the intracellular signalling and thus the ability of stem cells to be activated by BMP and its derived molecules. We have therefore identified a combination of bioactive polymers and BMP molecules that direct the stem cells towards bone forming cells.

Growth factor treatment to overcome Alzheimer's dysfunctional signaling.

The number of people suffering from Alzheimers disease (AD) will increase as the world population ages, creating a huge socio-economic burden. The three pathophysiological hallmarks of AD are the cholinergic system dysfunction, the β-amyloid peptide deposition and the Tau protein hyperphosphorylation. Current treatments have only transient effects and each tends to concentrate on a single pathophysiological aspect of AD. This review first provides an overall view of AD in terms of its pathophysiological symptoms and signaling dysfunction. We then examine the therapeutic potential of growth factors (GFs) by showing how they can overcome the dysfunctional cell signaling that occurs in AD. Finally, we discuss new alternatives to GFs that help overcome the problem of brain uptake, such as small peptides, with evidence from some of our unpublished data on human neuronal cell line.

Preventing Mek1 activation influences the responses of human osteosarcoma cells to bone morphogenetic proteins 2 and 9

It was recently suggested that bone morphogenetic protein (BMP)-2 may be useful for treating osteosarcoma cells. BMP-9, which has been patented to treat breast and prostate cancers, has a higher osteoinductive potential than BMP-2. Peptides derived from the knuckle epitope of BMPs (pBMPs) also induced osteogenic differentiation. However, the effect of BMP-9 and pBMPs on osteosarcoma cells is unclear. We analyzed the effects of BMP-2, BMP-9, pBMP-2, and pBMP-9 on the behavior of human MG-63 and SaOS-2 osteosarcoma cells. An inhibitor of MEK1 activation (PD98059) that prevents downstream extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and a specific inhibitor of p38 were also used as mitogen activated protein kinase-targeting therapy is being investigated as a treatment modality for osteosarcoma. BMP-2 and BMP-9 (1.92 nmol/l) induced the phosphorylation of Smad1/5/8 in both osteosarcoma cells within 1 h but had different effects on mitogen activated protein kinase pathways. Whereas BMP-2 mainly activated ERK1/2, BMP-9 phosphorylated p38 within 1 h. pBMP-2 did not activate either the Smad or ERK/p38, whereas pBMP-9, like BMP-9, induced both Smad1/5/8 and p38 phosphorylation. p38 activation by BMP-9 or pBMP-9 was also enhanced by PD98059. However, BMP-2 or BMP-9 increased the amounts of distal-less homeobox 5 and Osterix mRNAs in SaOS-2 cells within 6 h, whereas pBMP-9 had no effect. PD98059 promoted the highest level of Osterix mRNA in SaOS-2 cells incubated with BMP-2 or BMP-9, whereas p38 inhibitor had no effect. Furthermore, PD98059 induced the lowest proliferation of MG-63 cells incubated with BMP-2, whereas p38 inhibitor did not affect the proliferation of either osteosarcoma cell line. Therefore a combination of BMP-2 or BMP-9 and an inhibitor of MEK1 may be a promising tool for regulating osteosarcoma cell behavior.

The mixed-lineage kinase DLK undergoes Src-dependent tyrosine phosphorylation and activation in cells exposed to vanadate or platelet-derived growth factor (PDGF)

Some data in the literature suggest that serine/threonine phosphorylation is required for activation of the mixed-lineage kinases (MLKs), a subgroup of mitogen-activated protein kinase kinase kinases (MAPKKKs). In this report, we demonstrate that the MLK family member DLK is activated and concurrently tyrosine-phosphorylated in cells exposed to the protein tyrosine phosphatase inhibitor vanadate. Tyrosine phosphorylation appears crucial for activation as incubation of vanadate-activated DLK molecules with a tyrosine phosphatase substantially reduced DLK enzymatic activity. Interestingly, the effects of vanadate on DLK are completely blocked by treatment with a Src family kinase inhibitor, PP2, or the expression of short hairpin RNA (shRNA) directed against Src. DLK also fails to undergo vanadate-stimulated tyrosine phosphorylation and activation in fibroblasts which lack expression of Src, Yes and Fyn, but reintroduction of wild-type Src or Fyn followed by vanadate treatment restores this response. In addition to vanadate, stimulation of cells with platelet-derived growth factor (PDGF) also induces tyrosine phosphorylation and activation of DLK by a Src-dependent mechanism. DLK seems important for PDGF signaling because its depletion by RNA interference substantially reduces PDGF-stimulated ERK and Akt kinase activation. Thus, our findings suggest that Src-dependent tyrosine phosphorylation of DLK may be important for regulation of its activity, and they support a role for DLK in PDGF signaling.

Identification of a growth factor mimicking the synergistic effect of fetal bovine serum on BMP-9 cell response.

The bone morphogenetic proteins (BMPs) are potent osteogenic molecules that are used for bone repair in delivery systems and in regenerative medicine. We studied the responses of murine MC3T3-E1 preosteoblasts to doses of recombinant human (rh)BMP-9 with and without fetal bovine serum (FBS). rhBMP-2 was used as a control since it is currently approved by the Food and Drug Administration for bone application. We analyzed the major cell signaling pathways and the expression of osteogenic markers. Without FBS, BMP-9 had a similar effect on MC3T3-E1 preosteoblast differentiation in comparison to BMP-2. In contrast, FBS reduced the EC50 of BMP-9 fourfold to sixfold, as determined by osterix gene expression and alkaline phosphatase (ALP) activity, while it had no influence on EC50 of BMP-2. As suggested by MAPK inhibitor assays, FBS could induce an intracellular signaling environment that favors cell response to BMP-9 by inhibiting ERK1/2 activation and increasing p38 phosphorylation. Finally, IGF-2 (100 ng/mL) could mimic the effect of FBS on BMP-9 cell response in terms of MAPK signaling and ALP activity. Thus, the action of BMP-9 on preosteoblast differentiation can be greatly improved by IGF-2. This finding may well be critical for developing optimal growth factor delivery systems and bone tissue engineering strategies.