Characterizing expression pattern of Six2Cre during mouse craniofacial development.

Craniofacial development is a complex process involving diverse cell populations. Various transgenic Cre lines have been developed to facilitate studying gene function in specific tissues. In this study, we have characterized the expression pattern of Six2Cre mice at multiple stages during craniofacial development.

Characterizing the role of Pdgfra in calvarial development.

Background: Mammalian calvarium is composed of flat bones developed from two origins, neural crest, and mesoderm. Cells from both origins exhibit similar behavior but express distinct transcriptomes. It is intriguing to ask whether genes shared by both origins play similar or distinct roles in development.

Deregulated Rac1 Activity in Neural Crest Controls Cell Proliferation, Migration and Differentiation During Midbrain Development.

Mutations in allele are implicated in multiple brain tumors, indicating a rigorous control of Rac1 activity is required for neural tissue normal development and homeostasis. To understand how elevated Rac1 activity affects neural crest cells (NCCs) development, we have generated mice, in which a constitutively active Rac1 mutant is expressed specifically in NCCs derivatives. Our results revealed that augmented Rac1 activity leads to enlarged midbrain and altered cell density, accompanied by increased NCCs proliferation rate and misrouted cell migration.

Pdgfra regulates multipotent cell differentiation towards chondrocytes via inhibiting Wnt9a/beta-catenin pathway during chondrocranial cartilage development.

The mammalian skull is composed of the calvarial bones and cartilages. Malformation of craniofacial cartilage has been identified in multiple human syndromes. However, the mechanisms of their development remain largely unknown.

Expression pattern of Kmt2d in murine craniofacial tissues.

Formation of the calvaria is a multi-staged process and is regulated by multiple genetic factors. Disruption of normal calvarial development usually causes craniosynostosis, a prevalent birth defect characterized by premature fusion of calvarial bone. Recent studies have identified mutations of KMT2D allele in patients with craniosynostosis, indicating a potential role for Kmt2d in calvarial development.

Identification and characterization of the antiplasmodial activity of Hsp90 inhibitors.

Background: The recent reduction in mortality due to malaria is being threatened by the appearance of Plasmodium falciparum parasites that are resistant to artemisinin in Southeast Asia. To limit the impact of resistant parasites and their spread across the world, there is a need to validate anti-malarial drug targets and identify new leads that will serve as foundations for future drug development programmes targeting malaria. Towards that end, the antiplasmodial potential of several Hsp90 inhibitors was characterized.

Regulation of transforming growth factor-beta1 (TGF-β1)-induced pro-fibrotic activities by circadian clock gene BMAL1.

Background: BMAL1 is a transcriptional activator of the molecular clock feedback network. Besides its role in generating circadian rhythms, it has also been shown to be involved in the modulation of cell proliferation, autophagy and cancer cell invasion. However, the role of BMAL1 in pulmonary fibrogenesis is still largely unknown.

The mechanism and function of mitogen-activated protein kinase activation by ARF1.

Mitogen-activated protein kinases (MAPK) can be activated by a number of biochemical pathways through distinct signaling molecules. We have recently revealed a novel function for the Ras-like small GTPase ADP-ribosylation factor 1 (ARF1) in mediating the activation of Raf1-MEK-ERK1/2 pathway by G protein-coupled receptors [Dong C, Li C and Wu G (2011) J Biol Chem 286, 43,361-43,369]. Here, we have further defined the underlying mechanism and the possible function of ARF1-mediated MAPK pathway.

Deregulation of selective autophagy during aging and pulmonary fibrosis: the role of TGFβ1.

Aging constitutes a significant risk factor for fibrosis, and idiopathic pulmonary fibrosis (IPF) is characteristically associated with advancing age. We propose that age-dependent defects in the quality of protein and cellular organelle catabolism may be causally related to pulmonary fibrosis. Our research found that autophagy diminished with corresponding elevated levels of oxidized proteins and lipofuscin in response to lung injury in old mice and middle-aged mice compared to younger animals.

G-protein-coupled receptor interaction with small GTPases.

In addition to heterotrimeric G-proteins, Ras-like small GTPases are also involved in regulating physiological functions of the G-protein-coupled receptor (GPCR) superfamily. In particular, Rab and ARF GTPases function either as "traffic cops" to coordinate receptor targeting to specific locations or as "signal transducers" to directly mediate receptor signal propagation. As revealed in protein-protein interaction assays, GPCRs may use specific motifs to physically interact with small GTPases, providing important insights into the underlying molecular mechanisms.

A triple arg motif mediates α(2B)-adrenergic receptor interaction with Sec24C/D and export.

Recent studies have demonstrated that cargo exit from the endoplasmic reticulum (ER) may be directed by ER export motifs recognized by components of the coat protein II (COPII) vesicles. However, little is known about ER export motifs and vesicle targeting of the G protein-coupled receptor (GPCR) superfamily. Here, we have demonstrated that a triple Arg (3R) motif in the third intracellular loop functions as a novel ER export signal for α(2B)-adrenergic receptor (α(2B)-AR).

Regulation of α(2B)-adrenergic receptor-mediated extracellular signal-regulated kinase 1/2 (ERK1/2) activation by ADP-ribosylation factor 1.

A number of signaling molecules are involved in the activation of the mitogen-activated protein kinase (MAPK) pathway by G protein-coupled receptors. In this study, we have demonstrated that α(2B)-adrenergic receptor (α(2B)-AR) interacts with ADP-ribosylation factor 1 (ARF1), a small GTPase involved in vesicle-mediated trafficking, in an agonist activation-dependent manner and that the interaction is mediated through a unique double Trp motif in the third intracellular loop of the receptor. Interestingly, mutation of the double Trp motif and siRNA-mediated depletion of ARF1 attenuate α(2B)-AR-mediated activation of extracellular signal-regulated kinases 1/2 (ERK1/2) without altering receptor intracellular trafficking, whereas expression of the constitutively active mutant ARF1Q71L and ARNO, a GDP-GTP exchange factor of ARF1, markedly enhances the activation of Raf1, MEK1, and ERK1/2.

Inactive-state preassembly of G(q)-coupled receptors and G(q) heterotrimers.

G protein-coupled receptors (GPCRs) transmit signals by forming active-state complexes with heterotrimeric G proteins. It has been suggested that some GPCRs also assemble with G proteins before ligand-induced activation and that inactive-state preassembly facilitates rapid and specific G protein activation. However, no mechanism of preassembly has been described, and no functional consequences of preassembly have been demonstrated.

Di-acidic motifs in the membrane-distal C termini modulate the transport of angiotensin II receptors from the endoplasmic reticulum to the cell surface.

The molecular mechanisms underlying the endoplasmic reticulum (ER) export and cell surface transport of nascent G protein-coupled receptors (GPCRs) have just begun to be revealed and previous studies have shown that hydrophobic motifs in the putative amphipathic 8(th) α-helical region within the membrane-proximal C termini play an important role. In this study, we demonstrate that di-acidic motifs in the membrane-distal, nonstructural C-terminal portions are required for the exit from the ER and transport to the plasma membrane of angiotensin II receptors, but not adrenergic receptors. More interestingly, distinct di-acidic motifs dictate optimal export trafficking of different angiotensin II receptors and export ability of each acidic residue in the di-acidic motifs cannot be fully substituted by other acidic residue.

Alpha2B-adrenergic receptor interaction with tubulin controls its transport from the endoplasmic reticulum to the cell surface.

It is well recognized that the C terminus (CT) plays a crucial role in modulating G protein-coupled receptor (GPCR) transport from the endoplasmic reticulum (ER) to the cell surface. However the molecular mechanisms that govern CT-dependent ER export remain elusive. To address this issue, we used α(2B)-adrenergic receptor (α(2B)-AR) as a model GPCR to search for proteins interacting with the CT.

In vitro and in vivo antitumor activity of melatonin receptor agonists.

Melatonin has been shown to inhibit the proliferation of estrogen receptor α (ERα)-positive human breast cancer cells in vitro and suppress the growth of carcinogen-induced mammary tumors in rats. Melatonin's antiproliferative effect is mediated, at least in part, through the MT1 melatonin receptor and mechanisms involving modulation of the estrogen-signaling pathway. To develop melatonin analogs with greater therapeutic effects, we have examined the in vitro and in vivo antimitotic activity of two MT1/MT2 melatonin receptor agonists, S23219-1 and S23478-1.

Melatonin inhibits mitogenic cross-talk between retinoic acid-related orphan receptor alpha (RORalpha) and ERalpha in MCF-7 human breast cancer cells.

Luciferase reporter constructs and transient co-transfection approaches demonstrate that elevated expression of RORalpha1 augments 17-beta-estradiol (E(2))-induced transcriptional activation of the full-length ERalpha, but not truncated ERalpha constructs (ABCD or CDEF), in MCF-7 breast cancer and HEK293 embryonic kidney cells, and that physiologic concentrations of MLT inhibit the individual and combined transcriptional activity of ERalpha by RORalpha1 and E(2). Gel mobility shift and co-immunoprecipitation (IP)/pull-down assays demonstrate that RORalpha1 and ERalpha do not interact directly at the DNA-binding level or as heterodimers, however, RORalpha1 augments E(2)-induced pS2 and cyclin D1 mRNA expression while MLT inhibits RORalpha1/E(2)-induced expression of pS2 and cyclin D1 in MCF-7 cells.

Rab8 interacts with distinct motifs in alpha2B- and beta2-adrenergic receptors and differentially modulates their transport.

The molecular mechanism underlying the post-Golgi transport of G protein-coupled receptors (GPCRs) remains poorly understood. Here we determine the role of Rab8 GTPase, which modulates vesicular protein transport between the trans-Golgi network (TGN) and the plasma membrane, in the cell surface targeting of alpha(2B)- and beta(2)-adrenergic receptors (AR). Transient expression of GDP- and GTP-bound Rab8 mutants and short hairpin RNA-mediated knockdown of Rab8 more potently inhibited the cell surface expression of alpha(2B)-AR than beta(2)-AR.

ADP-ribosylation factors modulate the cell surface transport of G protein-coupled receptors.

ADP-ribosylation factors (ARFs) regulate vesicular traffic through recruiting coat proteins. However, their functions in the anterograde transport of nascent G protein-coupled receptors (GPCRs) from the endoplasmic reticulum to the plasma membrane remain poorly explored. Here we show that treatment with brefeldin A, an inhibitor of guanine nucleotide exchange on ARFs, markedly attenuated the cell surface numbers of alpha(2B)-adrenergic receptor (AR), beta(2)-AR, angiotensin II type 1 receptor, and chemokine (CXC motif) receptor 4.

A single conserved leucine residue on the first intracellular loop regulates ER export of G protein-coupled receptors.

The intrinsic structural determinants for export trafficking of G protein-coupled receptors (GPCRs) have been mainly identified in the termini of the receptors. In this report, we determined the role of the first intracellular loop (ICL1) in the transport from the endoplasmic reticulum (ER) to the cell surface of GPCRs. The alpha(2B)-adrenergic receptor (AR) mutant lacking the ICL1 is unable to traffic to the cell surface and to initiate signaling measured as ERK1/2 activation.

Anterograde trafficking of G protein-coupled receptors: function of the C-terminal F(X)6LL motif in export from the endoplasmic reticulum.

We have reported previously that the F(X)(6)LL motif in the C termini is essential for export of alpha(2B)-adrenergic (alpha(2B)-AR) and angiotensin II type 1 receptors (AT1Rs) from the endoplasmic reticulum (ER). Here, we further demonstrate that mutation of the F(X)(6)LL motif similarly abolished the cell-surface expression of alpha(2B)-AR, AT1R, alpha(1B)-AR, and beta(2)-AR, suggesting that the F(X)(6)LL motif plays a general role in ER export of G protein-coupled receptors (GPCRs). Mutation of Phe to Val, Leu, Trp, and Tyr, and mutation of LL to FF and VV, markedly inhibited alpha(2B)-AR transport, indicating that the F(X)(6)LL function cannot be fully substituted by other hydrophobic residues.

Alteration of the MT1 melatonin receptor gene and its expression in primary human breast tumors and breast cancer cell lines.

The MT1 melatonin receptor is bound and activated by the pineal hormone melatonin. This G protein-coupled melatonin receptor is expressed in human breast tumor cell lines, and when activated, mediates the growth-suppressive and steroid hormone/nuclear receptor modulatory actions of melatonin on breast tumor cells. In the current studies, we have examined the expression of the MT1 receptor in breast cancer cell lines and primary human breast tumors and correlated MT1 receptor expression with the deletion, rearrangement and amplification of the MT1 gene and established markers of breast cancer such as tumor size, stage, estrogen receptor alpha (ERalpha) and progesterone receptor (PR) expression.

The Galphai and Galphaq proteins mediate the effects of melatonin on steroid/thyroid hormone receptor transcriptional activity and breast cancer cell proliferation.

Melatonin, via its MT1 receptor, but not the MT2 receptor, can modulate the transcriptional activity of various nuclear receptors - estrogen receptor alpha (ERalpha) and retinoic acid receptor alpha (RARalpha), but not ERbeta- in MCF-7, T47D, and ZR-75-1 human breast cancer cell lines. The anti-proliferative and nuclear receptor modulatory actions of melatonin are mediated via the MT1 G protein-coupled receptor expressed in human breast cancer cells. However, the specific G proteins and associated pathways involved in the nuclear receptor transcriptional regulation by melatonin are not yet clear.

Regulation of anterograde transport of adrenergic and angiotensin II receptors by Rab2 and Rab6 GTPases.

Three Rab GTPases, Rab1, Rab2 and Rab6, are involved in protein transport between the endoplasmic reticulum (ER) and the Golgi. Whereas Rab1 regulates the anterograde ER-to-Golgi transport, Rab2 and Rab6 coordinate the retrograde Golgi-to-ER transport. We have previously demonstrated that Rab1 differentially modulates the export trafficking of distinct G protein-coupled receptors (GPCRs).