Fung-Fang Wang

Transcriptional upregulation of DDR2 by ATF4 facilitates osteoblastic differentiation through p38 MAPK-mediated Runx2 activation.

Deficiency of the collagen receptor discoidin domain receptor tyrosine kinase (DDR2) in mice and humans results in dwarfism and short limbs, of which the mechanism remains unknown. Here we report that DDR2 is a key regulator of osteoblast differentiation. DDR2 mRNA expression was increased at an early stage of induced osteoblast differentiation. In the subchondral bone of human osteoarthritic knee, DDR2 was detected in osteoblastic cells. In mouse embryos, DDR2 expression was found from E11 to E15, preceding osteocalcin (OCN) and coinciding with Runx2 expression. Activating transcription factor 4 (ATF4) enhanced DDR2 mRNA expression, and knockdown of ATF4 expression delayed DDR2 induction during osteoblast differentiation. A CCAAT/enhancer binding protein (C/EBP) binding site at −1150 bp in the DDR2 promoter was required for ATF4‐mediated DDR2 activation. C/EBPβ bound to and cooperated with ATF4 in stimulating DDR2 transcription; accordingly, the ATF4 mutants deficient of C/EBPβ binding were incapable of transactivating DDR2. Overexpression of DDR2 increased osteoblast‐specific gene expression. Conversely, knockdown of DDR2 suppressed osteogenic marker gene expression and matrix mineralization during the induced osteogenesis. The stimulation of p38 MAPK by DDR2 was required for DDR2‐induced activation of Runx2 and OCN promoters. Together our findings uncover a pathway in which ATF4, by binding to C/EBPβ transcriptionally upregulates DDR2 expression, and DDR2, in turn, activates Runx2 through p38 MAPK to promote osteoblast differentiation.

p53 downstream target DDA3 is a novel microtubule-associated protein that interacts with end-binding protein EB3 and activates β -catenin pathway

We have previously identified mouse DDA3 as a p53-inducible gene. To explore the functional role of DDA3, we screened a mouse brain cDNA library by the yeast two-hybrid assay, and identified the microtubule plus-end binding protein EB3 as a DDA3-interacting protein. Binding of DDA3 to EB3 was verified by glutathione S-transferase (GST) pull-down assay and subcellular colocalization; co-immunoprecipitation further indicated that interaction of these two proteins within cells required intact microtubules. Domains of DDA3-EB3 interaction were mapped by GST pull-down assay to amino acids 118–241 and 242–329 of DDA3 and the N- and C-termini of EB3. Immunofluorescence analysis revealed colocalization of DDA3 with microtubules in various cell phases, and regions encompassing aa 118–241 and 242–329 contained microtubule-interacting and bundling activities. In vitro microtubule-binding assay showed that DDA3 and EB3 associated directly with microtubules, and cooperated with each other for microtubule binding. In addition, DDA3 bound to the EB3 interacting partner adenomatous polyposis coli 2 (APC2), a homolog of the tumor suppressor APC, which is a component of the β-catenin destruction complex. Ectopic expression of DDA3 and EB3 enhanced β-catenin-dependent transactivation and cyclin D1 production, whereas knockdown of endogenous DDA3 or EB3 inhibited β-catenin-mediated transactivation and the ability of cells to form colonies. Together, our results identify DDA3 as a novel microtubule-associated protein that binds to EB3, and implicate DDA3 and EB3 in the β-catenin-mediated growth signaling.

Identification of a novel mouse p53 target gene DDA3

We have identified a novel p53 regulated gene designated DDA3 through differential mRNA display on IW32 erythroleukemia cells containing a temperature sensitive p53 allele, tsp53val-135. DDA3 mRNA induction could be observed in all sublines expressing tsp53val-135 cultured at permissive temperature as well as in NIH3T3 cells undergoing DNA damage. Upregulation of DDA3 could be detected within 2 h after down-shifting the temperature to 32.5°C; upon shifting back to 38.5°C, DDA3 mRNA rapidly degraded with a half-life of less than 2 h. Actinomycin D, but not cycloheximide, inhibited the p53 dependent DDA3 induction, suggesting that the activation is through transcriptional regulation and does not require de novo protein synthesis. DDA3 was expressed in multiple mouse tissues including brain, spleen, lung, kidney and testis. Full-length DDA3 cDNA was cloned and it contained an open reading frame predicted to encode a proline rich protein of 329 amino acids. Overexpression of DDA3 in H1299 lung carcinoma cells suppressed colony formation. These results suggest that DDA3 is a p53-regulated gene that might participate in the p53-mediated growth suppression.

The Prosurvival Activity of p53 Protects Cells from UV-Induced Apoptosis by Inhibiting c-Jun NH2-terminal Kinase Activity and Mitochondrial Death Signaling

The cytoprotective function of p53 recently has been exploited as a therapeutic advantage for cancer prevention; agents activating the prosurvival activity of p53 are shown to prevent UV-induced damages. To explore the mechanisms of p53-mediated protection from UV-induced apoptosis, we have established stable clones of H1299 lung carcinoma cells expressing a temperature-sensitive p53 mutant, tsp53V143A. At the permissive temperature of 32°C, the tsp53V143A-expressing cells were arrested in G1 phase without the occurrence of apoptosis; consistent with this is the preferential induction of genes related to growth arrest and DNA damage repair. Previous expression of functional tsp53V143A for ≥18 hours inhibited the release of proapoptotic molecules from mitochondria and protected the cells from UV-induced apoptosis; moreover, it suppressed the activation of c-Jun NH2-terminal kinase (JNK) signaling and relieved the effect of UV on p53 target gene activation. p53 associated with JNK and inhibited its kinase activity. Using the p53-null H1299 cells, we showed that inhibition of JNK blocked the UV-elicited mitochondrial death signaling and caspase activation. Our results suggest that the ability of p53 to bind and inactivate JNK, together with the activation of the p53 target genes related to cell cycle arrest and DNA damage repair, is responsible for its protection of cells against UV-induced apoptosis.

Induction of fos and sis proto-oncogenes and genes of the extracellular matrix proteins during butyrate induced glioma differentiation

Sodium butyrate has been shown to inhibit the growth and induce the differentiation of F-98 rat glioma cells. In agreement with the morphological changes, we have found that mRNAs for fibronectin and collagen in these cells could be reversibly induced by butyrate. While Ki-ras mRNA levels remained relatively unchanged, mRNAs for fos and sis increased significantly during the course of butyrate induced differentiation. c-fos induction can be detected 30 min after butyrate addition, a peak level (greater than 20 fold) was reached at 2 h, with a subsequent gradual decline. c-sis induction was detectable 24 h after butyrate exposure, at which time the cells have assumed morphological transition. Interestingly, the sis mRNA induction was not reversible upon butyrate withdrawal. The sis mRNA half-life increased from 40 min in the untreated cells to 100 min in the butyrate induced cells indicating that the increase in the stability of sis mRNA contributed, at least in part, to the elevated levels of sis expression. These findings demonstrate a coordinated induction of fibronectin and collagen genes in the butyrate-treated F-98 cells. In addition, fos and sis transcripts were differentially induced; a rapid and transient induction of fos followed by an irreversible induction of sis at a later stage of differentiation.

Human DDA3 is an oncoprotein down-regulated by p53 and DNA damage.

Mouse DDA3 (mDDA3) is a microtubule-associated protein that promotes cell growth. mDDA3 contains an intronic p53 binding motif that is absent in human DDA3 (hDDA3), and is transcriptionally activated during DNA damage in a p53-dependent way. We now report that hDDA3 mRNA and protein levels were suppressed by p53, as well as in DNA damaged cells harboring wild type, but not mutant-p53 expression. We have located three consensus El-Deiry decamers at -1478/-1403 of the hDDA3 gene, and shown by chromatin immunoprecipitation that p53 bound to the region. Luciferase analysis showed that the hDDA3 promoter containing the putative p53 binding motif was responsible for p53-mediated repression. Expression of hDDA3 decreased the cells requirement for serum, furthermore, overexpression of hDDA3 mRNA was detected in hepatoma tissues. Together our results show that hDDA3 is a p53- and DNA-damage down-regulated target that exhibits oncogenic characteristics.

Rad is a p53 direct transcriptional target that inhibits cell migration and is frequently silenced in lung carcinoma cells

The p53 tumor suppressor exerts its function mainly as a transcriptional activator. Here we show that the Ras-related small GTPase Rad, an inhibitor of Rho kinase, is a direct transcriptional target of p53. Expression of Rad messenger RNA (mRNA) and protein was induced by DNA damage in a p53-dependent manner. The −2934/−2905-bp Rad promoter region, to which p53 bound, was required for p53-mediated Rad gene activation. Treatment by DNA damaging agents increased p53 occupancy and histone acetylation in the region of Rad promoter containing the p53-binding site. Expression of Rad diminished the inhibitory phosphorylation at Ser3 of cofilin, a regulator of actin dynamics, and suppressed migration and invasiveness of cancer cells. Knockdown of Rad promoted cell migration and alleviated the p53-mediated migration suppression. Frequent loss of Rad mRNA and protein expression was observed in non-small cell lung carcinoma tissues. Together our results reveal a mechanism that p53 may inhibit cell migration by disrupting actin dynamics via Rad activation and implicate a tumor suppressor role of Rad in lung cancer.

Mouse DDA3 gene is a direct transcriptional target of p53 and p73

The p53 tumor suppressor is a transcription factor that activates the expression of many target genes. We have previously reported the identification of a p53-regulated mouse gene DDA3. The 5′ upstream genomic region of the mouse DDA3 was cloned, and sequence analysis revealed the presence of a potential p53 response element (RE2) residing at nucleotides +390∼+409 relative to the first translation start site. When fused upstream to a luciferase reporter gene, 5′ genomic regions of the DDA3 gene containing RE2 were shown to be responsive to the wild-type, but not mutant p53, in a transient transfection assay. RE2 was sufficient to confer the transactivation responsiveness to p53. Furthermore, gel mobility shift analysis showed that RE2 formed specific complexes with wild-type p53. Induction of DDA3 was found in adriamycin treated normal mouse embryonic fibroblast cells (MEF), but not in p53 knockout (p53−/−) MEF. Overexpression of p73 induced DDA3 mRNA expression, and luciferase reporter analysis indicated that RE2 was responsive to transactivation by members of the p73 family proteins. Consistent with these findings, elevated expression of p73 protein and DDA3 mRNA were observed concomitantly in the p53−/− MEF cells treated with cisplatin. These results together demonstrated that DDA3 is a transcriptional target gene of p53 and its related-protein p73.

Differential Effect of Age on Transforming Growth Factor-β1 Inhibition of Prolactin Gene Expression Versus Secretion in Rat Anterior Pituitary Cells1

Transforming growth factor-β1 (TGF-β1) synthesized in the pituitary may act as an autocrine/paracrine regulator of lactotrope function. We examined the effects of TGF-β1 on PRL messenger RNA (mRNA), PRL synthesis, and PRL secretion in cultured anterior pituitary (AP) cells from rats at different ages. APs excised from ovariectomized female Sprague-Dawley rats, either young (2–3 months old; average serum PRL: 9 ng/ml), middle-aged (11–12 months old; average serum PRL: 133 ng/ml), or old (24 months old; average serum PRL: 159 ng/ml), were dispersed and cultured for 5 days. Then, cells were washed and challenged with increasing doses of TGF-β1 (0–100 ng/ml) for 1–48 h in serum-free medium. Northern blot analysis showed an increase in basal PRL mRNA levels, and a decrease in responsiveness to TGF-β1 with age. TGF-β1 suppressed PRL mRNA in a dose- and time-dependent manner in cells from young rats. Maximum inhibition was observed at 0.5–1 ng/ml of TGF-β1. At 0.5 ng/ml TGF-β1, significant reduction in PRL mRNA ...

p53 Target DDA3 binds ASPP2 and inhibits its stimulation on p53-mediated BAX activation

The p53 tumor suppressor functions in maintaining the integrity of the genome. We have previously reported that DDA3 is an oncoprotein transcriptionally regulated by p53. To explore mechanisms underlying DDA3 action, we searched for its interacting proteins by yeast two-hybrid screening, and identified ASPP2, a p53 binding protein, as its binding partner. The DDA3/ASPP2 binding was confirmed in vitro by GST pull-down and in vivo by immunofluorescence assay, which indicated colocalization of DDA3 and ASPP2. Interacting domain of DDA3 was mapped to amino acids 118-241, whereas both the N- and C-terminal regions of ASPP2 were capable of binding to DDA3. DDA3 dose-dependently inhibited ASPP2 in stimulating the p53-mediated BAX promoter activation without interfering the binding of ASPP2 to p53. Together these results identify ASPP2 as a bona fide DDA3 interacting protein, and suggest that the ASPP2/DDA3 interaction may inhibit ASPP2 in stimulating the apoptotic signaling of p53.