Microphthalmia Gene Product as a Signal Transducer in cAMP-Induced Differentiation of Melanocytes

Melanocyte differentiation characterized by an increased melanogenesis, is stimulated by α-melanocyte–stimulating hormone through activation of the cAMP pathway. During this process, the expression of tyrosinase, the enzyme that controls melanin synthesis is upregulated. We previously showed that cAMP regulates transcription of the tyrosinase gene through a CATGTG motif that binds microphthalmia a transcription factor involved in melanocyte survival. Further, microphthalmia stimulates the transcriptional activity of the tyrosinase promoter and cAMP increases the binding of microphthalmia to the CATGTG motif. These observations led us to hypothesize that microphthalmia mediates the effect of cAMP on the expression of tyrosinase. The present study was designed to elucidate the mechanism by which cAMP regulates microphthalmia function and to prove our former hypothesis, suggesting that microphthalmia is a key component in cAMP-induced melanogenesis. First, we showed that cAMP upregulates the transcription of microphthalmia gene through a classical cAMP response element that is functional only in melanocytes. Then, using a dominant-negative mutant of microphthalmia, we demonstrated that microphthalmia is required for the cAMP effect on tyrosinase promoter. These findings disclose the mechanism by which cAMP stimulates tyrosinase expression and melanogenesis and emphasize the critical role of microphthalmia as signal transducer in cAMP-induced melanogenesis and pigment cell differentiation.

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Abstract 5304: Dominant Negative Suppression of Rad Leads to Intracellular Ca 2+ Overload via Up-Regulation of Cardiac Ryanodine Receptor Activity

Circulation ◽

10.1161/circ.118.suppl_18.s_523-b ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

Mitsushige Murata ◽

Hirotaka Yada ◽

Hiroyuki Yamakawa ◽

Yoshiyasu Aizawa ◽

Shinsuke Yuasa ◽

...

Keyword(s):

Ryanodine Receptor ◽

Critical Role ◽

Mean Amplitude ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Release Channel ◽

Intracellular Ca ◽

Mouse Cells ◽

Cardiac Ryanodine Receptor

Background: We have recently reported that the ras-related small G-protein Rad plays a critical role in generating arrhythmias via regulation of L-type Ca 2+ channel. However, it has remained unclear whether or not the mechanism for its arrhythmogenesis is attributed only to L-type Ca 2+ channel activity. This study was designed to demonstrate the role of Rad in intracellular calcium homeostasis by cardiac-specific dominant negative suppression of Rad. Methods and results: Transgenic mice that overexpress dominant negative mutant Rad (DN Rad TG) driven by α-myosin heavy chain promoter were generated. To measure intracellular Ca 2+ concentration ([Ca 2+ ] i ), we recorded [Ca 2+ ] i transients and Ca 2+ sparks from isolated cardiomyocytes by confocal microscope. The mean amplitude of [Ca 2+ ] i transient was significantly increased in DN Rad TG cardiomyocytes, compared with WT littermate mouse cells (F/F 0 3.3 ± 0.2, n = 25, in DN Rad TG cells vs. 2.4 ± 0.1, n = 30, in WT cells, P<0.05). The frequency of Ca 2+ sparks was significantly higher in TG cells than in WT cells (5.2 ± 0.6 sparks•100 μm −1 •s −1 , n = 45 in TG cells vs. 1.9 ± 0.3 sparks•100 μm −1 •s −1 , n = 45, in WT controls, P<0.05), although there were no significant differences in their amplitudes. Furthermore, SR Ca 2+ content was not altered in TG cells, as assessed by caffeine-induced [Ca 2+ ] i transient. These results suggested that the properties of fundamental SR Ca 2+ release units might have been affected by the inhibition of endogenous Rad activity. To elucidate the mechanism for the increased frequency of SR Ca 2+ release channel, phosphorylation of Ser 2809 on cardiac ryanodine receptor (RYR2) was examined. The phosphorylation of RYR2 at Ser 2809 was significantly enhanced in TG mouse hearts compared with WT mice, implicating the upregulation of RYR2 activity in TG mice. Additionally, this Rad-mediated phosphorylation of RYR2 was regulated by protein kinase A (PKA) activity. Conclusions: Our results provided the first evidence that Rad regulated RYR2 activity via PKA signaling pathway. Thus, Rad might play a critical role in cellular Ca 2+ homeostasis via its inhibitory effects on RYR2 as well as L-type Ca 2+ channel.

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Exogenous C2-ceramide activates c-fos serum response element via Rac-dependent signalling pathway

Biochemical Journal ◽

10.1042/bj3301009 ◽

1998 ◽

Vol 330 (2) ◽

pp. 1009-1014 ◽

Cited By ~ 18

Author(s):

Byung-Chul KIM ◽

Jae-Hong KIM

Keyword(s):

Phospholipase A2 ◽

Reporter Gene ◽

Critical Role ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Dependent Manner ◽

Response Element ◽

Serum Response ◽

Negative Mutant

Ceramide is an important regulatory molecule implicated in a variety of biological processes in response to stress and cytokines. To understand the signal transduction pathway of ceramide to the nucleus, in the present study, we examined whether C2-ceramide, a cell permeable ceramide, activates c-fos serum response element (SRE). Treatment of Rat-2 fibroblast cells with C2-ceramide caused the stimulation of c-fos SRE-dependent reporter gene activity in a dose- and time-dependent manner by transient transfection analysis. Next, we examined the role of Rho family GTPases in the ceramide-induced signalling to SRE activation. By reporter gene analysis following transient transfections with various plasmids expressing a dominant negative mutant form of Cdc42, Rac1 or RhoA, C2-ceramide-induced SRE activation was shown to be selectively repressed by pEXV-RacN17 encoding a dominant negative mutant of Rac1, suggesting that Rac activity is essential for the signalling cascade of ceramide to the nucleus. In a further study to analyse the downstream mediator of Rac in the ceramide-signalling pathway, we observed that either pretreatment with mepacrine, a potent and specific inhibitor of phospholipase A2, or co-transfection with antisense cytosolic phospholipase A2 (cPLA2) oligonucleotide repressed the C2-ceramide-induced SRE activation selectively, implying a critical role of cPLA2 in C2-ceramide-induced signalling to nucleus. Consistent with these results, the translocation of cPLA2 protein as well as the release of arachidonic acid, a principal product of phospholipase A2, was rapidly induced by the addition of C2-ceramide in a Rac-dependent manner. Together, our findings suggest the critical role of ‘Rac and subsequent activation of phospholipase A2’ in ceramide-signalling to nucleus.

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Atypical Protein Kinase C Is Involved in the Evolutionarily Conserved Par Protein Complex and Plays a Critical Role in Establishing Epithelia-Specific Junctional Structures

The Journal of Cell Biology ◽

10.1083/jcb.152.6.1183 ◽

2001 ◽

Vol 152 (6) ◽

pp. 1183-1196 ◽

Cited By ~ 292

Author(s):

Atsushi Suzuki ◽

Tomoyuki Yamanaka ◽

Tomonori Hirose ◽

Naoyuki Manabe ◽

Keiko Mizuno ◽

...

Keyword(s):

Epithelial Cells ◽

Cell Polarity ◽

Protein Complex ◽

Mdck Cells ◽

Critical Role ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Surface Polarity ◽

C Elegans ◽

Evolutionarily Conserved

We have previously shown that during early Caenorhabditis elegans embryogenesis PKC-3, a C. elegans atypical PKC (aPKC), plays critical roles in the establishment of cell polarity required for subsequent asymmetric cleavage by interacting with PAR-3 [Tabuse, Y., Y. Izumi, F. Piano, K.J. Kemphues, J. Miwa, and S. Ohno. 1998. Development (Camb.). 125:3607–3614]. Together with the fact that aPKC and a mammalian PAR-3 homologue, aPKC-specific interacting protein (ASIP), colocalize at the tight junctions of polarized epithelial cells (Izumi, Y., H. Hirose, Y. Tamai, S.-I. Hirai, Y. Nagashima, T. Fujimoto, Y. Tabuse, K.J. Kemphues, and S. Ohno. 1998. J. Cell Biol. 143:95–106), this suggests a ubiquitous role for aPKC in establishing cell polarity in multicellular organisms. Here, we show that the overexpression of a dominant-negative mutant of aPKC (aPKCkn) in MDCK II cells causes mislocalization of ASIP/PAR-3. Immunocytochemical analyses, as well as measurements of paracellular diffusion of ions or nonionic solutes, demonstrate that the biogenesis of the tight junction structure itself is severely affected in aPKCkn-expressing cells. Furthermore, these cells show increased interdomain diffusion of fluorescent lipid and disruption of the polarized distribution of Na+,K+-ATPase, suggesting that epithelial cell surface polarity is severely impaired in these cells. On the other hand, we also found that aPKC associates not only with ASIP/PAR-3, but also with a mammalian homologue of C. elegans PAR-6 (mPAR-6), and thereby mediates the formation of an aPKC-ASIP/PAR-3–PAR-6 ternary complex that localizes to the apical junctional region of MDCK cells. These results indicate that aPKC is involved in the evolutionarily conserved PAR protein complex, and plays critical roles in the development of the junctional structures and apico-basal polarization of mammalian epithelial cells.

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Prevention of TNF-α-induced apoptosis in polyamine-depleted IEC-6 cells is mediated through the activation of ERK1/2

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00342.2003 ◽

2004 ◽

Vol 286 (3) ◽

pp. G479-G490 ◽

Cited By ~ 36

Author(s):

Sujoy Bhattacharya ◽

Ramesh M. Ray ◽

Leonard R. Johnson

Keyword(s):

Epithelial Cells ◽

Cytochrome C ◽

Critical Role ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Cytochrome C Release ◽

Erk Phosphorylation ◽

Tnf Α ◽

Induced Apoptosis ◽

Jnk Activation

It has been documented that polyamines play a critical role in the regulation of apoptosis in intestinal epithelial cells. We have recently reported that protection from TNF-α/cycloheximide (CHX)-induced apoptosis in epithelial cells depleted of polyamines is mediated through the inactivation of a proapoptotic mediator, JNK. In this study, we addressed the involvement of the MAPK pathway in the regulation of apoptosis after polyamine depletion of IEC-6 cells. Polyamine depletion by α-difluromethylornithine (DFMO) resulted in the sustained activation of ERK in response to TNF-α/CHX treatment. Pretreatment of polyamine-depleted IEC-6 cells with a cell membrane-permeable MEK1/2 inhibitor, U-0126, significantly inhibited TNF-α/CHX-induced ERK phosphorylation and significantly increased DNA fragmentation, JNK activity, and caspase-3 activity in response to TNF-α/CHX. Moreover, the dose dependency of U-0126-mediated inhibition of TNF-α/ CHX-induced ERK phosphorylation correlated with the reversal of the antiapoptotic effect of DFMO. IEC-6 cells expressing constitutively active MEK1 had decreased TNF-α/CHX-induced JNK phosphorylation and were significantly protected from apoptosis. Conversely, a dominant-negative MEK1 resulted in high basal activation of JNK, cytochrome c release, and spontaneous apoptosis. Polyamine depletion of the dominant-negative MEK1 cells did not prevent JNK activation or cytochrome c release and failed to confer protection from both TNF-α/CHX and camptothecin-induced apoptosis. Finally, expression of a dominant-negative mutant of JNK significantly protected IEC-6 cells from TNF-α/CHX-induced apoptosis. These data indicate that polyamine depletion results in the activation of ERK, which inhibits JNK activation and protects cells from apoptosis.

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Knockout of inositol-requiring enzyme 1α in pro-opiomelanocortin neurons decreases fat mass via increasing energy expenditure

Open Biology ◽

10.1098/rsob.160131 ◽

2016 ◽

Vol 6 (8) ◽

pp. 160131 ◽

Cited By ~ 9

Author(s):

Yuzhong Xiao ◽

Tingting Xia ◽

Junjie Yu ◽

Yalan Deng ◽

Hao Liu ◽

...

Keyword(s):

Energy Expenditure ◽

Metabolic Diseases ◽

Critical Role ◽

Liver Steatosis ◽

Leptin Resistance ◽

Hormone Production ◽

Melanocyte Stimulating Hormone ◽

Diet Induced Obesity ◽

Brown Adipose

Although numerous functions of inositol-requiring enzyme 1α (IRE1α) have been identified, a role of IRE1α in pro-opiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus is largely unknown. Here, we showed that mice lacking IRE1α specifically in POMC neurons (PIKO) are lean and resistant to high-fat diet-induced obesity and obesity-related insulin resistance, liver steatosis and leptin resistance. Furthermore, PIKO mice had higher energy expenditure, probably due to increased thermogenesis in brown adipose tissue. Additionally, α-melanocyte-stimulating hormone production was increased in the hypothalamus of PIKO mice. These results demonstrate that IRE1α in POMC neurons plays a critical role in the regulation of obesity and obesity-related metabolic disorders. Our results also suggest that IRE1α is not only an endoplasmic reticulum stress sensor, but also a new potential therapeutic target for obesity and obesity-related metabolic diseases.

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Centrosome cohesion is regulated by a balance of kinase and phosphatase activities

Journal of Cell Science ◽

10.1242/jcs.114.20.3749 ◽

2001 ◽

Vol 114 (20) ◽

pp. 3749-3757 ◽

Cited By ~ 6

Author(s):

Patrick Meraldi ◽

Erich A. Nigg

Keyword(s):

Protein Phosphatase ◽

Kinase Activity ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Microtubule Depolymerization ◽

Drug Induced ◽

Microtubule Network ◽

Similar Phenotype ◽

Underlying Mechanisms

Centrosome cohesion and separation are regulated throughout the cell cycle, but the underlying mechanisms are not well understood. Since overexpression of a protein kinase, Nek2, is able to trigger centrosome splitting (the separation of parental centrioles), we have surveyed a panel of centrosome-associated kinases for their ability to induce a similar phenotype. Cdk2, in association with either cyclin A or E, was as effective as Nek2, but several other kinases tested did not significantly interfere with centrosome cohesion. Centrosome splitting could also be triggered by inhibition of phosphatases, and protein phosphatase 1α (PP1α) was identified as a likely physiological antagonist of Nek2. Furthermore, we have revisited the role of the microtubule network in the control of centrosome cohesion. We could confirm that microtubule depolymerization by nocodazole causes centrosome splitting. Surprisingly, however, this drug-induced splitting also required kinase activity and could specifically be suppressed by a dominant-negative mutant of Nek2. These studies highlight the importance of protein phosphorylation in the control of centrosome cohesion, and they point to Nek2 and PP1α as critical regulators of centrosome structure.

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β1-Integrin and PI 3-kinase regulate RhoA-dependent activation of skeletal α-actin promoter in myoblasts

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.2000.278.6.h1736 ◽

2000 ◽

Vol 278 (6) ◽

pp. H1736-H1743 ◽

Cited By ~ 23

Author(s):

Lei Wei ◽

Wei Zhou ◽

Lu Wang ◽

Robert J. Schwartz

Keyword(s):

Gene Expression ◽

Promoter Activity ◽

Serum Response Factor ◽

Critical Role ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Muscle Gene Expression ◽

Actin Promoter ◽

Muscle Gene ◽

Negative Mutant

RhoA GTPase, a regulator of actin cytoskeleton, is also involved in regulating c- fos gene expression through its effect on serum response factor (SRF) transcriptional activity. We have also shown that RhoA plays a critical role in myogenesis and regulates expression of SRF-dependent muscle genes, including skeletal α-actin. In the present study, we examined whether the RhoA signaling pathway cross talks with other myogenic signaling pathways to modulate skeletal α-actin promoter activity in myoblasts. We found that extracellular matrix proteins and the β1-integrin stimulated RhoA-dependent activation of the α-actin promoter. The muscle-specific isoform β1Dselectively activated the α-actin promoter in concert with RhoA but inhibited the c- fos promoter. In addition, focal adhesion kinase (FAK) and phosphatidylinositol (PI) 3-kinase were required for full activation of the α-actin promoter by RhoA. Expression of a dominant negative mutant of FAK, application of wortmannin to cultured myoblasts, or expression of a dominant negative mutant of PI 3-kinase inhibited α-actin promoter activity induced by RhoA. These results suggest that RhoA, β1-integrin, FAK, and PI 3-kinase serve together as an important signaling network in regulating muscle gene expression.

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MITOCHONDRIA AS REVERSIBLE REGULATORS OF AGING ASSOCIATED SKIN WRINKLES AND HAIR LOSS IN MICE

Innovation in Aging ◽

10.1093/geroni/igz038.1461 ◽

2019 ◽

Vol 3 (Supplement_1) ◽

pp. S395-S395

Author(s):

Keshav K Singh

Keyword(s):

Mouse Model ◽

Hair Loss ◽

Transgene Expression ◽

Skin Aging ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Dominant Negative Mutation ◽

Polymerase Domain ◽

Skin Wrinkles

Abstract To evaluate the consequences of the decline in mtDNA content associated with aging we have created an inducible mouse model expressing, in the polymerase domain of POLG1, a dominant-negative mutation that induces depletion of mtDNA. We utilized this inducible mouse model to modulate mitochondrial function by depleting and repleting the mtDNA content. We demonstrate that, in mice, ubiquitous expression of dominant-negative mutant POLG1 leads to 1) reduction of mtDNA content in skin, 2) skin wrinkles, and 3) hair loss. By turning off the mutant POLG1 transgene expression in the whole animal, the skin and hair phenotypes revert to normal after repletion of mtDNA. Thus, we have developed whole-animal mtDNA depleter-repleter mice. These mice present evidence that mtDNA homeostasis is involved in skin aging phenotype and loss of hair and provide an unprecedented opportunity to create tissue-specific mitochondrial modulation to determine the role of the mitochondria in a particular tissue.

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Phospholipase D1 mediates bFGF-induced Bcl-2 expression leading to neurite outgrowth in H19-7 cells

Biochemical Journal ◽

10.1042/bj20110302 ◽

2011 ◽

Vol 441 (1) ◽

pp. 407-416 ◽

Cited By ~ 8

Author(s):

Sung Nyo Yoon ◽

Kang Sik Kim ◽

Ju Hwan Cho ◽

Weina Ma ◽

Hye-Jin Choi ◽

...

Keyword(s):

Neurite Outgrowth ◽

Dominant Negative ◽

Signal Transducer ◽

Downstream Effector ◽

Phospholipase D1 ◽

Protein Kinase Cα ◽

Phosphoinositide 3 Kinase ◽

Interfering Rna ◽

Transcription 3

The purpose of the present study was to investigate the role of PLD (phospholipase D) in bFGF (basic fibroblast growth factor)-induced Bcl-2 expression and to examine whether overexpressed Bcl-2 influences neurite outgrowth in immortalized hippocampal progenitor cells (H19-7 cells). We found that Bcl-2 expression was maximally induced by bFGF within 24 h, and that this effect was reduced by inhibiting PLD1 expression with PLD1 small interfering RNA or by overexpressing DN (dominant-negative)-PLD1, whereas PLD1 overexpression markedly induced Bcl-2 expression. bFGF treatment activated Ras, Src, PI3K (phosphoinositide 3-kinase), PLCγ (phospholipase Cγ) and PKCα (protein kinase Cα). Among these molecules, Src and PKCα were not required for Bcl-2 expression. PLD activity was decreased by Ras, PI3K or PLCγ inhibitor, suggesting that PLD1 activation occurred through Ras, PI3K or PLCγ. We found that Ras was the most upstream molecule among these proteins, followed by the PI3K/PLCγ pathway, indicating that bFGF-induced PLD activation took place through the Ras/PI3K/PLCγ pathway. Furthermore, PLD1 was required for activation of JNK (c-Jun N-terminal kinase), which led to activation of STAT3 (signal transducer and activator of transcription 3) and finally Bcl-2 expression. When Bcl-2 was overexpressed, neurite outgrowth was stimulated along with induction of neurotrophic factors such as brain-derived neurotrophic factor and neurotrophin 4/5. In conclusion, PLD1 acts as a downstream effector of bFGF/Ras/PI3K/PLCγ signalling and regulates Bcl-2 expression through JNK/STAT3, which leads to neurite outgrowth in H19-7 cells.

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Rottlerin Reduces cAMP/CREB-Mediated Melanogenesis via Regulation of Autophagy

International Journal of Molecular Sciences ◽

10.3390/ijms20092081 ◽

2019 ◽

Vol 20 (9) ◽

pp. 2081 ◽

Cited By ~ 7

Author(s):

Nurinanda Prisky Qomaladewi ◽

Mi-Yeon Kim ◽

Jae Youl Cho

Keyword(s):

Signaling Pathway ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Camp Response Element Binding ◽

Sequential Process ◽

Camp Response Element ◽

Melanin Production ◽

Melanocyte Stimulating Hormone ◽

Cellular Autophagy

Melanogenesis is the sequential process of melanin production by melanocytes in order to protect the skin from harmful stimuli. Melanogenesis is disrupted by radiation exposure, which results in the differentiation of melanocytes into melanoma. Recently, some methods have been developed to maintain the instability of melanogenesis in melanoma by activating cellular autophagy. However, there is still a lack of knowledge about how autophagy is involved in the regulation of melanogenesis in melanoma cells. Here, we used rottlerin as an autophagy inducer to investigate the role of the cyclic adenosine monophosphate (cAMP)/cAMP response element binding (CREB) signaling pathway in melanogenesis. We found that rottlerin can inhibit melanin production by targeting cAMP, which is initially activated by alpha-melanocyte stimulating hormone (α-MSH). Our findings suggest that rottlerin has a pivotal role as an autophagy inducer in the regulation of melanogenesis by targeting the cAMP/CREB signaling pathway.

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