Protein kinase D as a potential new target for cancer therapy

Protein kinase D is a novel family of serine/threonine kinases and diacylglycerol receptors that belongs to the calcium/calmodulin-dependent kinase superfamily. Evidence has established that specific PKD isoforms are dysregulated in several cancer types, and PKD involvement has been documented in a variety of cellular processes important to cancer development, including cell growth, apoptosis, motility, and angiogenesis. In light of this, there has been a recent surge in the development of novel chemical inhibitors of PKD. This review focuses on the potential of PKD as a chemotherapeutic target in cancer treatment and highlights important recent advances in the development of PKD inhibitors.     

Identification of a novel phosphorylation site in c-jun directly targeted in vitro by protein kinase D

Protein kinase D (PKD) phosphorylates the c-jun amino-terminal in vitro at site(s) distinct from JNK [C. Hurd, R.T. Waldron, E. Rozengurt, Protein kinase D complexes with c-jun N-terminal kinase via activation loop phosphorylation and phosphorylates the c-jun N-terminus, Oncogene 21 (2002) 2154-2160], but the sites have not been identified. Here, metabolic (32)P-labeling of c-jun protein in COS-7 cells indicated that PKD phosphorylates c-jun in vivo at a site(s) between aa 43-93, a region containing important functional elements. On this basis, the PKD-mediated phosphorylation site(s) was further characterized in vitro using GST-c-jun fusion proteins. PKD did not incorporate phosphate into Ser63 and Ser73, the JNK sites in GST-c-jun(1-89). Rather, PKD and JNK could sequentially phosphorylate distinct site(s) simultaneously. By mass spectrometry of tryptic phosphopeptides, Ser58 interposed between the JNK-binding portion of the delta domain and the adjacent TAD1 was identified as a prominent site phosphorylated in vitro by PKD. These data were further supported by kinase reactions using truncations or point-mutations of GST-c-jun. Together, these data suggest that PKD-mediated phosphorylation modulates c-jun at the level of its N-terminal functional domains.     

Expression of cGMP-dependent protein kinase type I in mature white adipocytes

The presence of cGMP-dependent protein kinase I (cGKI) in murine adipocytes has been questioned, although cGKI was implicated in the thermogenic program of fat cells (FCs) and to exert anti-hypertrophic/-inflammatory effects in white adipose tissue. Herein, cGKI was detected in adipocytes from control mice, whereas FCs from global cGKI knockouts (cGKI^−/−) and cGKIα rescue (αRM) mice remained cGKI-negative. cGKI mutants exhibit decreased adipocyte size, plasma leptin levels and reduced body-weights as compared to litter-matched controls. Low abundance of adiponectin in WAT and plasma of αRM animals together with previously confirmed high IL-6 levels indicate a low-grade inflammation. However, αRMs were protected from streptozotocin-induced hyperglycemia. Our results suggest that cGMP/cGKI affects both glucose and FC homeostasis in more complex mode than previously thought.     

Troglitazone activates TRPV1 and causes deacetylation of PPARγ in 3T3-L1 cells

Published research suggests that activation of transient receptor potential vanilloid subfamily 1 (TRPV1) enhances the expression and deacetylation of peroxisome proliferator-activated receptor gamma (PPARγ) to cause browning of white adipose tissue. Here, we show that TRPV1 activation by capsaicin significantly prevents high fat diet-induced obesity in mice. This is associated with an increase in the expression and deacetylation of PPARγ in the epididymal fat of these mice. Consistent with the TRPV1 activation in vivo, overexpression of TRPV1 enhanced the PPARγ and other thermogenic genes in cultured 3T3-L1 preadipocytes. To determine the interaction between TRPV1 and PPARγ signaling, we analyzed the effect of Troglitazone (Trog; a thiazolidinedione derivative and an agonist of PAARγ) treatment on cultured 3T3-L1 cells. Trog enhanced the expression of TRPV1, PPARγ and thermogenic proteins in undifferentiated 3T3-L1 cells but not in differentiated cells. Acute application of Trog stimulated a robust Ca²⁺ influx into 3T3-L1 cells and TRPV1 inhibition by capsazepine prevented this. More interestingly, Trog or capsaicin treatment caused the deacetylation of PPARγ in 3T3-L1 cells and inhibition of TRPV1 or Sirtuin 1 - prevented this. Our data suggest a novel effect of Trog to induce PPARγ deacetylation by activating TRPV1. This research has a significant implication on the role of TRPV1 and PPARγ signaling in the browning of white adipose tissue.     

PI3K/Akt is involved in brown adipogenesis mediated by growth differentiation factor-5 in association with activation of the Smad pathway

We have previously demonstrated promotion by growth differentiation factor-5 (GDF5) of brown adipogenesis for systemic energy expenditure through a mechanism relevant to activating the bone morphological protein (BMP) receptor/mothers against decapentaplegic homolog (Smad)/peroxisome proliferator-activated receptor gamma co-activator 1α (PGC-1α) pathway. Here, we show the involvement of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in brown adipogenesis mediated by GDF5. Overexpression of GDF5 in cells expressing adipocyte protein-2 markedly accelerated the phosphorylation of Smad1/5/8 and Akt in white and brown adipose tissues. In brown adipose tissue from heterozygous GDF5^Rgsc451 mutant mice expressing a dominant-negative (DN) GDF5 under obesogenic conditions, the basal phosphorylation of Smad1/5/8 and Akt was significantly attenuated. Exposure to GDF5 not only promoted the phosphorylation of both Smad1/5/8 and Akt in cultured brown pre-adipocytes, but also up-regulated Pgc1a and uncoupling protein-1 expression in a manner sensitive to the PI3K/Akt inhibitor Ly294002 as well as retroviral infection with DN-Akt. GDF5 drastically promoted BMP-responsive luciferase reporter activity in a Ly294002-sensitive fashion. Both Ly294002 and DN-Akt markedly inhibited phosphorylation of Smad5 in the nuclei of brown pre-adipocytes. These results suggest that PI3K/Akt signals play a role in the GDF5-mediated brown adipogenesis through a mechanism related to activation of the Smad pathway.     

Involvement of AMP-activated protein kinase in fat depot-specific metabolic changes during starvation

The mechanisms controlling fat depot-specific metabolism are poorly understood. During starvation of mice, downregulation of lipogenic genes, suppression of fatty acid synthesis, and increases in lipid oxidation were all more pronounced in epididymal than in subcutaneous fat. In epididymal fat, relatively strong upregulation of uncoupling protein 2 and phosphoenolpyruvate carboxykinase genes was found. In mice maintained both at 20 and 30 degrees C, AMP-activated protein kinase was activated in epididymal but did not change in subcutaneous fat. Our results suggest that AMPK may have a role in the different response of various fat depots to starvation.     

The C-terminal 26-residue peptide of serpin A1 stimulates proliferation of breast and liver cancer cells: role of protein kinase C and CD47

C26, the C-terminal 26 residue peptide of serpin A1, significantly increased cell proliferation in cultures of hepatoma cells, but not in porcine kidney epithelial cells, human skin fibroblasts or keratinocytes. The mitogenic activity of C26 was preferentially inhibited with a protein kinase C (PKC) inhibitor, an antibody against CD47 and CD47 short interfering RNA. The mutant C26-K19R,N22M, imitating a thrombospondin-like cell adhesion motif, increased the mitogenic activity in both Hep G2 cells and MCF-7 breast cancer cells. Phosphorylation of C26 at T24 (a putative PKC phosphorylation site) resulted in a 1.9-2.5 increase in mitogenic activity over C26 in MCF-7 cells.     

Ceramide 1-phosphate/ceramide, a switch between life and death

Ceramide is a well-characterized sphingolipid metabolite and second messenger that participates in numerous biological processes. In addition to serving as a precursor to complex sphingolipids, ceramide is a potent signaling molecule capable of regulating vital cellular functions. Perhaps its major role in signal transduction is to induce cell cycle arrest, and promote apoptosis. In contrast, little is known about the metabolic or signaling pathways that are regulated by the phosphorylated form of ceramide. It was first demonstrated that ceramide-1-phosphate (C1P) had mitogenic properties, and more recently it has been described as potent inhibitor of apoptosis and inducer of cell survival. C1P and ceramide are antagonistic molecules that can be interconverted in cells by kinase and phosphatase activities. An appropriate balance between the levels of these two metabolites seems to be crucial for cell and tissue homeostasis. Switching this balance towards accumulation of one or the other may result in metabolic dysfunction, or disease. Therefore, the activity of the enzymes that are involved in C1P and ceramide metabolism must be efficiently coordinated to ensure normal cell functioning.     

Selective modulation of the CAP/Cbl pathway in the adipose tissue of high fat diet treated rats

A high-fat diet (HFD) is associated with reduced glucose uptake in muscle, but not in adipose tissue. In the present study, we investigated whether a HFD can modulate glucose uptake in adipose tissue by increasing signal transduction through the CAP/Cbl pathway, independently of the PI3-K/Akt pathway. Our results suggest that, in HFD, the differential regulation of insulin-induced glucose uptake between skeletal muscle and adipose tissue may, in part, be a consequence of the CAP/Cbl/C3G pathway, since the expression of CAP and Cbl, and also the activation of this pathway were increased in adipose tissue but not in muscle.     

TNF-alpha and adipocyte biology

Dyslipidemia and insulin resistance are commonly associated with catabolic or lipodystrophic conditions (such as cancer and sepsis) and with pathological states of nutritional overload (such as obesity-related type 2 diabetes). Two common features of these metabolic disorders are adipose tissue dysfunction and elevated levels of tumour necrosis factor-alpha (TNF-alpha). Herein, we review the multiple actions of this pro-inflammatory adipokine on adipose tissue biology. These include inhibition of carbohydrate metabolism, lipogenesis, adipogenesis and thermogenesis and stimulation of lipolysis. TNF-alpha can also impact the endocrine functions of adipose tissue. Taken together, TNF-alpha contributes to metabolic dysregulation by impairing both adipose tissue function and its ability to store excess fuel. The molecular mechanisms that underlie these actions are discussed.     

Endoplasmic reticulum: ER stress regulates mitochondrial bioenergetics

Endoplasmic reticulum (ER) stress activates an adaptive unfolded protein response (UPR) that facilitates cellular repair, however, under prolonged ER stress, the UPR can ultimately trigger apoptosis thereby terminating damaged cells. The molecular mechanisms responsible for execution of the cell death program are relatively well characterized, but the metabolic events taking place during the adaptive phase of ER stress remain largely undefined. Here we discuss emerging evidence regarding the metabolic changes that occur during the onset of ER stress and how ER influences mitochondrial function through mechanisms involving calcium transfer, thereby facilitating cellular adaptation. Finally, we highlight how dysregulation of ER-mitochondrial calcium homeostasis during prolonged ER stress is emerging as a novel mechanism implicated in the onset of metabolic disorders.     

Live visualization and quantification of pathway signaling with dual fluorescent and bioluminescent reporters

Despite their fundamental importance, the dynamics of signaling pathways in living cells remain challenging to study, due to a lack of non-invasive tools for temporal assessment of signal transduction in desired cell models. Here we report a dual-reporter strategy that enables researchers to monitor signal transduction in mammalian cells in real-time, both temporally and quantitatively. This is achieved by co-expressing green fluorescent protein and firefly luciferase in response to signaling stimuli. To display the versatility of this approach, we constructed and assessed eight unique signaling pathway reporters. We further validated the system by establishing stable NF-κB pathway reporter cell lines. Using these stable cell lines, we monitored the activity of NF-κB-mediated inflammatory pathway in real-time, both visually and quantitatively. Live visualization has the power to reveal individual cell responses and is compatible with single cell analysis, In addition, we provide evidence that this system is readily amenable to a high-throughput format. Together, our findings demonstrate the potential of the dual reporter system, which significantly improves the capacity to study signal transduction pathways in mammalian cells.     

Neuroendocrine and metabolic activities of ghrelin gene products

Acylated ghrelin (AG) is a 28 amino acid gastric peptide a natural ligand for the growth hormone secretagogue (GHS) receptor type 1a (GHS-R1a), endowed with GH-secreting and orexigenic properties. Besides, ghrelin exerts several peripheral metabolic actions, including modulation of glucose homeostasis and stimulation of adipogenesis. Notably, AG administration causes hyperglycemia in rodents as in humans. Ghrelin pleiotropy is supported by a widespread expression of the ghrelin gene, of GHS-R1a and other unknown ghrelin binding sites. The existence of alternative receptors for AG, of several natural ligands for GHS-R1a and of acylation-independent ghrelin non-neuroendocrine activities, suggests that there might be a complex ‘ghrelin system’ not yet completely explored. Moreover, the patho-physiological implications of unacylated ghrelin (UAG), and obestatin (Ob), the other two ghrelin gene-derived peptides, need to be clarified. Within the next few years, we may better understand the ‘ghrelin system’, where we might envisage clinical applications.     

Genistein stimulates fatty acid oxidation in a leptin receptor-independent manner through the JAK2-mediated phosphorylation and activation of AMPK in skeletal muscle

Obesity is a public health problem that contributes to the development of insulin resistance, which is associated with an excessive accumulation of lipids in skeletal muscle tissue. There is evidence that soy protein can decrease the ectopic accumulation of lipids and improves insulin sensitivity; however, it is unknown whether soy isoflavones, particularly genistein, can stimulate fatty acid oxidation in the skeletal muscle. Thus, we studied the mechanism by which genistein stimulates fatty acid oxidation in the skeletal muscle. We showed that genistein induced the expression of genes of fatty acid oxidation in the skeletal muscle of Zucker fa/fa rats and in leptin receptor (ObR)-silenced C2C12 myotubes through AMPK phosphorylation. Furthermore, the genistein-mediated AMPK phosphorylation occurred via JAK2, which was possibly activated through a mechanism that involved cAMP. Additionally, the genistein-mediated induction of fatty acid oxidation genes involved PGC1α and PPARδ. As a result, we observed that genistein increased fatty acid oxidation in both the control and silenced C2C12 myotubes, as well as a decrease in the RER in mice, suggesting that genistein can be used in strategies to decrease lipid accumulation in the skeletal muscle.