Huazhang Guo

Phospholipase D2-dependent Inhibition of the Nuclear Hormone Receptor PPARγ by Cyclic Phosphatidic Acid

Cyclic phosphatidic acid (1-acyl-2,3-cyclic-glycerophosphate, CPA), one of natures simplest phospholipids, is found in cells from slime mold to humans and has a largely unknown function. We find here that CPA is generated in mammalian cells in a stimulus-coupled manner by phospholipase D2 (PLD2) and binds to and inhibits the nuclear hormone receptor PPARgamma with nanomolar affinity and high specificity through stabilizing its interaction with the corepressor SMRT. CPA production inhibits the PPARgamma target-gene transcription that normally drives adipocytic differentiation of 3T3-L1 cells, lipid accumulation in RAW264.7 cells and primary mouse macrophages, and arterial wall remodeling in a rat model in vivo. Inhibition of PLD2 by shRNA, a dominant-negative mutant, or a small molecule inhibitor blocks CPA production and relieves PPARgamma inhibition. We conclude that CPA is a second messenger and a physiological inhibitor of PPARgamma, revealing that PPARgamma is regulated by endogenous agonists as well as by antagonists.

Lysophosphatidic acid 2 receptor-mediated supramolecular complex formation regulates its antiapoptotic effect.

The G protein-coupled lysophosphatidic acid 2 (LPA2) receptor elicits prosurvival responses to prevent and rescue cells from apoptosis. However, G protein-coupled signals are not sufficient for the full protective effect of LPA2. LPA2 differs from other LPA receptor subtypes in the C-terminal tail, where it contains a zinc finger-binding motif for the interactions with LIM domain-containing TRIP6 and proapoptotic Siva-1, and a PDZ-binding motif through which it complexes with the NHERF2 scaffold protein. In this report, we identify a unique CXXC motif of LPA2 responsible for the binding to TRIP6 and Siva-1, and demonstrate that disruption of these macromolecular complexes or knockdown of TRIP6 or NHERF2 expression attenuates LPA2-mediated protection from chemotherapeutic agent-induced apoptosis. In contrast, knockdown of Siva-1 expression enhances this effect. Furthermore, a PDZ-mediated direct interaction between TRIP6 and NHERF2 facilitates their interaction with LPA2. Together, these results suggest that in addition to G protein-activated signals, the cooperation embedded in the LPA2-TRIP6-NHERF2 ternary complex provides a novel ligand-dependent signal amplification mechanism that is required for LPA2-mediated full activation of antiapoptotic signaling.

Lysophosphatidic acid-induced arterial wall remodeling: Requirement of PPARγ but not LPA1 or LPA2 GPCR

Lysophosphatidic acid (LPA) and its ether analog alkyl-glycerophosphate (AGP) elicit arterial wall remodeling when applied intralumenally into the uninjured carotid artery. LPA is the ligand of eight GPCRs and the peroxisome proliferator-activated receptor gamma (PPARgamma). We pursued a gene knockout strategy to identify the LPA receptor subtypes necessary for the neointimal response in a non-injury model of carotid remodeling and also compared the effects of AGP and the PPARgamma agonist rosiglitazone (ROSI) on balloon injury-elicited neointima development. In the balloon injury model AGP significantly increased neointima; however, rosiglitazone application attenuated it. AGP and ROSI were also applied intralumenally for 1h without injury into the carotid arteries of LPA(1), LPA(2), LPA(1&2) double knockout, and Mx1Cre-inducible conditional PPARgamma knockout mice targeted to vascular smooth muscle cells, macrophages, and endothelial cells. The neointima was quantified and also stained for CD31, CD68, CD11b, and alpha-smooth muscle actin markers. In LPA(1), LPA(2), LPA(1&2) GPCR knockout, Mx1Cre transgenic, PPARgamma(fl/-), and uninduced Mx1CrexPPARgamma(fl/-) mice AGP- and ROSI-elicited neointima was indistinguishable in its progression and cytological features from that of WT C57BL/6 mice. In PPARgamma(-/-) knockout mice, generated by activation of Mx1Cre-mediated recombination, AGP and ROSI failed to elicit neointima and vascular wall remodeling. Our findings point to a difference in the effects of AGP and ROSI between the balloon injury- and the non-injury chemically-induced neointima. The present data provide genetic evidence for the requirement of PPARgamma in AGP- and ROSI-elicited neointimal thickening in the non-injury model and reveal that the overwhelming majority of the cells in the neointimal layer express alpha-smooth muscle actin.

The early- and late stages in phenotypic modulation of vascular smooth muscle cells: differential roles for lysophosphatidic acid.

Lysophosphatidic acid (LPA) has been implicated as causative in phenotypic modulation (PM) of cultured vascular smooth muscle cells (VSMC) in their transition to the dedifferentiated phenotype. We evaluated the contribution of the three major LPA receptors, LPA1 and LPA2 GPCR and PPARgamma, on PM of VSMC. Expression of differentiated VSMC-specific marker genes, including smooth muscle alpha-actin, smooth muscle myosin heavy chain, calponin, SM-22alpha, and h-caldesmon, was measured by quantitative real-time PCR in VSMC cultures and aortic rings kept in serum-free chemically defined medium or serum- or LPA-containing medium using wild-type C57BL/6, LPA1, LPA2, and LPA1&2 receptor knockout mice. Within hours after cells were deprived of physiological cues, the expression of VSMC marker genes, regardless of genotype, rapidly decreased. This early PM was neither prevented by IGF-I, inhibitors of p38, ERK1/2, or PPARgamma nor significantly accelerated by LPA or serum. To elucidate the mechanism of PM in vivo, carotid artery ligation with/without replacement of blood with Krebs solution was used to evaluate contributions of blood flow and pressure. Early PM in the common carotid was induced by depressurization regardless of the presence/absence of blood, but eliminating blood flow while maintaining blood pressure or after sham surgery elicited no early PM. The present results indicate that LPA, serum, dissociation of VSMC, IGF-I, p38, ERK1/2, LPA1, and LPA2 are not causative factors of early PM of VSMC. Tensile stress generated by blood pressure may be the fundamental signal maintaining the fully differentiated phenotype of VSMC.

Basic helix–loop–helix protein E47-mediated p21Waf1/Cip1 gene expression regulates apoptosis of intestinal epithelial cells

Inhibition of ornithine decarboxylase by DFMO (alpha-difluromethylornithine) and subsequent polyamine depletion increases p21Cip1 protein, induces cell cycle arrest and confers resistance to apoptosis on intestinal epithelial cells. However, the mechanism by which polyamines regulate p21Cip1 expression and apoptosis is unknown. On the basis of the involvement of p21Cip1 as an anti-apoptotic protein, we tested the role of p21Cip1 in providing protection from apoptosis. Simultaneously, we investigated the role of E47, a basic helix-loop-helix protein, in the regulation of p21Cip1 gene transcription. Gene-specific siRNA (small interfering RNA) decreased E47 protein levels, increased p21Cip1 promoter activity and protein levels and protected cells from TNFalpha (tumour necrosis factor alpha)-induced apoptosis. Knockdown of p21Cip1 protein by siRNA resulted in cells becoming more susceptible to apoptosis. In contrast, incubation with EGF (epidermal growth factor) stimulated p21Cip1 mRNA and protein levels and rescued cells from apoptosis. During apoptosis, the level of E47 mRNA increased, causing a concomitant decrease in p21Cip1 mRNA and protein levels. Polyamine depletion decreased E47 mRNA levels and cell survival. Caspase 3-mediated cleavage of p130Cas has been implicated in p21Cip1 transcription. The progression of apoptosis led to a caspase 3-dependent cleavage of p130Cas and generated a 31 kDa fragment, which translocated to the nucleus, associated with nuclear E47 and inhibited p21Cip1 transcription. Polyamine depletion inhibited all these effects. Transient expression of the 31 kDa fragment prevented the expression of p21Cip1 protein and increased apoptosis. These results implicate p21Cip1 as an anti-apoptotic protein and suggest a role for polyamines in the regulation of p21Cip1 via the transcription repressor E47. Caspase-mediated cleavage of p130Cas generates a 31 kDa fragment, inhibits p21Cip1 transcription and acts as an amplifier of apoptotic signalling.

A two-step approach to qPCR experimental design and software for data analysis

Materials and methods For screening, a single biological sample is used for each control and treatment group. If screening shows interesting findings, confirmation follows with more biological replicates and a definitive statistical analysis using pooled data from the screening and confirmation steps. This experimental design for qPCR reduces reagent cost and labor without sacrificing sensitivity. To quantitate gene expression, we selected the comparative Ct method due to its simplicity, intuitiveness, and popularity. For statistical analysis, however, this two-step approach raised an interesting question; which parameter should be used for statistical analysis of the data from the screening step. With a single biological sample, the statistical analysis relies on the technical replicates of qPCR. Even though it is natural to assume a normal distribution of the relative gene expression levels of biological replicates based on the central limit theorem, it is unknown for technical replicates because the Ct values of the technical replicates have been non-linearly transformed to obtain relative gene expression levels. Hence, we studied the distribution of delta-Ct and relative gene expression levels from our qPCR data and found that the distribution of delta-Ct is symmetrical and approximately normal while the distribution of relative gene expression levels is skewed. Therefore, we chose deltaCt as our parameter for statistical analysis of the technical replicates from the screening step. To automate the data analysis of the screening step, we developed REALPLOT. A text file is used to enter Ct values and gene/ group information into REALPLOT that calculates relative gene expression, performs statistical analysis, and graphically displays the screening step data. To automate the data analysis after the confirmation step, we developed REALPOOL that pools the data from both the screening and confirmation steps, performs the final statistical analysis, and generates a publishable graphical representation. REALPLOT and REALPOOL were developed using the open-source statistical R environment. They are compatible with all major computer operating systems.

Clinical report of congenital lymphatic malformations and partial gigantism of the hands associated with a heterogeneous karyotype

Partial gigantism of the hands is a component of Proteus syndrome (OMIM 176920) [Wiedemann et al., 1983], Klippel– Trenaunay syndrome (OMIM 149000) [Cohen, 2000], and other disorders. One cause of partial gigantism can be congenital lymphatic malformations, which are in turn characterized by cystic dilatation of the lymphatic vessels [Greenlee et al., 1993; Fonkalsrud, 1994]. We recently observed a 45-year-old woman with partial gigantism of the hands and multiple lymphatic malformations. At birth, her hands were enlarged. The right index finger was amputated because it impaired hand function when she was 5years-old. Other congenital anomalies included subcutaneous masses in the left axillary fossa and on her back. The masses grew as the patient grew, and had unexplained intermitting disappearances and reappearances. At 45-years-old, the patient was admitted to a hospital because of colporrhagia of 3 months duration and was diagnosed with atypical endometrial hyperplasia and splenomegaly. She underwent a hysterectomy and splenectomy. There is no family history of either lymphatic malformations or gigantism of the hands. The patient’s facial features were normal. Partial gigantism of the hands was present (Fig. 1) with many overgrown phalanges of her hands in X-ray images (Fig. 2). Multiple masses were palpable in left axillary fossa and in the back and abdomen. Computed tomography and ultrasound showed multiple cystic masses in the liver, spleen, left axillary fossa (Fig. 3), right upper clavicle, mediastinum, bilateral ovaries, and subcutaneous tissue of her back. Pathologic analysis showed that the spleen was 40 19 12 cm, consisting of large cysts with associated multiple small subcapsular cysts containing clear, yellow fluid. Histology showed the cysts of spleen were lined by endothelium, the walls of the cysts consisted of smooth muscle cells and collagen. Fluorescence in situ hybridization (FISH) and spectral karyotyping (SKY) was performed as described earlier [Padilla-Nash et al., 2001]. The histological features of the splenic cysts were consistent with dilated lymphatic channels. SKY of peripheral blood leukocytes revealed a composite karyotype (Fig. 4A), mos 46,XX[12]/46,XX,ish del(17)(p12p13)(TP53)[5]/47,XX,þX[2]. FISH using a TP53-specific gene probe for the 17p13.1 locus and a whole chromosome paint for X was performed to further investigate the del(17) and to verify an extra copy of X as seen in SKY. Eleven cells were analyzed: five cells (46%) demonstrated two normal TP53 signals; four

RhoA activation stimulates IEC-6 cell proliferation by decreasing p21Wafl1/Cip1 and increasing Cdk2 expression in a polyamine dependent manner

Background & Aims: Although RhoA plays an important role m ceil proliferation and in Ras transformation in fibroblasts and mammary epithelial cells; its role in intestinal epithelial cells is still unknown. In our previous study, we showed that polyamine depletion (DFMO treatment) strongly inhibits proliferation of intestinal epithelial cells (IEC-6). In this report, we examined the effects of active RhoA and the mechanism underlying 1EC-6 cell proliferation, contact inhibition, and focus formation. We also examined whether polyamine depletion inhibits cell proliferation m the presence of constitutively active RhoA. Methods: Constitutively active RhoA and vector transfected 1EC-6 cell lines were used. Cells were grown in the presence or absence of DFMO, an irreversible inhibitor of omithine decarboxylase (ODC). Growth characteristics were assessed by microscopic observation, cell counting, and ceil cycle analysis by flow cytometry. Cell cycle regulators were evaluated by western blot, functional assay, and real-time PCR. Results: RhoA transfection significantly increased the rate of cell proliferation. These cells also demonstrated attenuated contact inhibition and conspicuous loci when they were fully confluent, increased proliferation was accompanied by decreased p21Wafl/Cipl, increased Cdk2 expression and activity. The inhibition of p21Wafl/Cipl was independent of p53. There was no activation of the Ras-Raf-Mek-Erk pathway in the RhoA transfected cell line. Polyamine depletion totally prevented the effect of activated RhoA on 1EC-6 cell proliferation and focus formation, decreased Cdk2 expression and activity independent of p21Wafl/Cipl. Conclusions: First, RhoA activation decreases p21, increase~ Cdk2 protein and Cdk2 activity, leading to the stimulation of intestinal epithelial cell proliferation and transformation; second, polyamine depletion totally prevents stimulating effect of RhoA on proliferation by decreasing Cdk2 expression and activity.