Sumana Datta

Arabidopsis vegetative storage protein is an anti-insect acid phosphatase

Indirect evidence previously suggested that Arabidopsis (Arabidopsis thaliana) vegetative storage protein (VSP) could play a role in defense against herbivorous insects. To test this hypothesis, other AtVSP-like sequences in Arabidopsis were identified through a Basic Local Alignment Search Tool search, and their transcriptional profiles were investigated. In response to methyl jasmonate application or phosphate starvation, AtVSP and AtVSP-like genes exhibited differential expression patterns, suggesting distinct roles played by each member. Arabidopsis VSP2 (AtVSP2), a gene induced by wounding, methyl jasmonate, insect feeding, and phosphate deprivation, was selected for bacterial expression and functional characterization. The recombinant protein exhibited a divalent cation-dependent phosphatase activity in the acid pH range. When incorporated into the diets of three coleopteran and dipteran insects that have acidic gut lumen, recombinant AtVSP2 significantly delayed development of the insects and increased their mortality. To further determine the biochemical basis of the anti-insect activity of the protein, the nucleophilic aspartic acid-119 residue at the conserved DXDXT signature motif was substituted by glutamic acid via site-directed mutagenesis. This single-amino acid alteration did not compromise the proteins secondary or tertiary structure, but resulted in complete loss of its acid phosphatase activity as well as its anti-insect activity. Collectively, we conclude that AtVSP2 is an anti-insect protein and that its defense function is correlated with its acid phosphatase activity.

Drosophila Perlecan modulates FGF and Hedgehog signals to activate neural stem cell division

Mutations in the Drosophila trol gene cause cell cycle arrest of neuroblasts in the larval brain. Here, we show that trol encodes the Drosophila homolog of Perlecan and regulates neuroblast division by modulating both FGF and Hh signaling. Addition of human FGF-2 to trol mutant brains in culture rescues the trol proliferation phenotype, while addition of a MAPK inhibitor causes cell cycle arrest of the regulated neuroblasts in wildtype brains. Like FGF, Hh activates stem cell division in the larval brain in a Trol-dependent fashion. Coimmunoprecipitation studies are consistent with interactions between Trol and Hh and between mammalian Perlecan and Shh that are not competed with heparin sulfate. Finally, analyses of mutations in trol, hh, and ttv suggest that Trol affects Hh movement. These results indicate that Trol can mediate signaling through both of the FGF and Hedgehog pathways to control the onset of stem cell proliferation in the developing nervous system.

Sonic Hedgehog signaling in advanced prostate cancer.

Abstract.The Hedgehog family of growth factors activate a highly conserved signaling system for cell-cell communication that regulates cell proliferation and differentiation during development. Abnormal activation of the Hedgehog pathway has been demonstrated in a variety of human tumors, including those of the skin, brain, lung and digestive tract. Hedgehog pathway activity in these tumors is required for cancer cell proliferation and tumor growth. Recent studies have uncovered the role for Hedgehog signaling in advanced prostate cancer and demonstrated that autocrine signaling by tumor cells is required for proliferation, viability, and invasive behavior. The level of Hedgehog activity correlates with the severity of the tumor and is both necessary and sufficient for metastatic behavior. Blockade of Hedgehog signaling leads to tumor shrinkage and remission in preclinical tumor xenograft models. Thus, Hedgehog signaling represents a novel pathway in prostate cancer that offers opportunities for prognostic biomarker development, drug targeting and therapeutic response monitoring.

Perlecan, a candidate gene for the CAPB locus, regulates prostate cancer cell growth via the Sonic Hedgehog pathway

BackgroundGenetic studies associated the CAPB locus with familial risk of brain and prostate cancers. We have identified HSPG2 (Perlecan) as a candidate gene for CAPB. Previously we have linked Perlecan to Hedgehog signaling in Drosophila. More recently, we have demonstrated the importance of Hedgehog signaling in humans for advanced prostate cancer.ResultsHere we demonstrate Perlecan expression in prostate cancer, and its function in prostate cancer cell growth through interaction and modulation of Sonic Hedgehog (SHH) signaling. Perlecan expression in prostate cancer tissues correlates with a high Gleason score and rapid cell proliferation. Perlecan is highly expressed in prostate cancer cell lines, including androgen insensitive cell lines and cell lines selected for metastatic properties. Inhibition of Perlecan expression in these cell lines decreases cell growth. Simultaneous blockade of Perlecan expression and androgen signaling in the androgen-sensitive cell line LNCaP was additive, indicating the independence of these two pathways. Perlecan expression correlates with SHH in tumor tissue microarrays and increased tumor cell proliferation based on Ki-67 immunohistochemistry. Inhibition of Perlecan expression by siRNA in prostate cancer cell lines decreases SHH signaling while expression of the downstream SHH effector GLI1 rescues the proliferation defect. Perlecan forms complexes with increasing amounts of SHH that correlate with increasing metastatic potential of the prostate cancer cell line. SHH signaling also increases in the more metastatic cell lines. Metastatic prostate cancer cell lines grown under serum-starved conditions (low androgen and growth factors) resulted in maintenance of Perlecan expression. Under low androgen, low growth factor conditions, Perlecan expression level correlates with the ability of the cells to maintain SHH signaling.ConclusionWe have demonstrated that Perlecan, a candidate gene for the CAPB locus, is a new component of the SHH pathway in prostate tumors and works independently of androgen signaling. In metastatic tumor cells increased SHH signaling correlates with the maintenance of Perlecan expression and more Perlecan-SHH complexes. Perlecan is a proteoglycan that regulates extracellular and stromal accessibility to growth factors such as SHH, thus allowing for the maintenance of SHH signaling under growth factor limiting conditions. This proteoglycan represents an important central regulator of SHH activity and presents an ideal drug target for blocking SHH effects.

The Drosophila Perlecan gene trol regulates multiple signaling pathways in different developmental contexts

BackgroundHeparan sulfate proteoglycans modulate signaling by a variety of growth factors. The mammalian proteoglycan Perlecan binds and regulates signaling by Sonic Hedgehog, Fibroblast Growth Factors (FGFs), Vascular Endothelial Growth Factor (VEGF) and Platelet Derived Growth Factor (PDGF), among others, in contexts ranging from angiogenesis and cardiovascular development to cancer progression. The Drosophila Perlecan homolog trol has been shown to regulate the activity of Hedgehog and Branchless (an FGF homolog) to control the onset of stem cell proliferation in the developing brain during first instar. Here we extend analysis of trol mutant phenotypes to show that trol is required for a variety of developmental events and modulates signaling by multiple growth factors in different situations.ResultsDifferent mutations in trol allow developmental progression to varying extents, suggesting that trol is involved in multiple cell-fate and patterning decisions. Analysis of the initiation of neuroblast proliferation at second instar demonstrated that trol regulates this event by modulating signaling by Hedgehog and Branchless, as it does during first instar. Trol protein is distributed over the surface of the larval brain, near the regulated neuroblasts that reside on the cortical surface. Mutations in trol also decrease the number of circulating plasmatocytes. This is likely to be due to decreased expression of pointed, the response gene for VEGF/PDGF signaling that is required for plasmatocyte proliferation. Trol is found on plasmatocytes, where it could regulate VEGF/PDGF signaling. Finally, we show that in second instar brains but not third instar brain lobes and eye discs, mutations in trol affect signaling by Decapentaplegic (a Transforming Growth Factor family member), Wingless (a Wnt growth factor) and Hedgehog.ConclusionThese studies extend the known functions of the Drosophila Perlecan homolog trol in both developmental and signaling contexts. These studies also highlight the fact that Trol function is not dedicated to a single molecular mechanism, but is capable of regulating different growth factor pathways depending on the cell-type and event underway.

Expression of cyclin E or DP/E2F rescues the G1 arrest of trol mutant neuroblasts in the Drosophila larval central nervous system.

The trol locus of Drosophila regulates the timing of neuroblast proliferation. In trol mutants, quiescent neuroblasts fail to begin division. We have investigated this cell cycle arrest to examine trol function. Induced expression of cyclin E or DP/E2F in trol mutants results in normal levels of dividing neuroblasts, while cyclin B expression has no effect. cyclin E expression is lower in the trol mutant larval CNS as assayed by quantitative RT-PCR, suggesting that trol neuroblasts are arrested in G1 due to lack of Cyclin E. Neither cyclin E nor E2F expression can phenocopy ana mutations, indicating that arrest caused by lack of Trol is different from Ana-mediated arrest.

Activation of neuroblast proliferation in explant culture of the Drosophila larval CNS

The developmental control of neuroblast proliferation is absolutely required for the assembly and function of the central nervous system. A lethal mutation in trol results in the failure of quiescent neuroblasts to begin division at the appropriate time. I have established a culture system in which quiescent neuroblasts in explants of Drosophila larval CNSs initiate cell division in vitro to normal in vivo levels. This activation requires removal of the CNS for culture after a specific developmental stage and the presence of fetal calf serum or a larval extract in the medium. Either supplement is effective when heat-treated. Substitution of the steroid hormone ecdysone or the non-steroidal ecdysone analog RH5992 for either fetal calf serum or larval extract also results in activation of neuroblast proliferation. Culture of trolsd CNSs with wildtype larval extract or ecdysone results in the defective neuroblast proliferation phenotype observed in trol mutants in vivo, while culture of wildtype CNSs with trolsd extract produces normal neuroblast proliferation.

Branchless and Hedgehog operate in a positive feedback loop to regulate the initiation of neuroblast division in the Drosophila larval brain

The Drosophila central nervous system is produced by two rounds of neurogenesis: one during embryogenesis to form the larval brain and one during larval stages to form the adult central nervous system. Neurogenesis caused by the activation of neural stem division in the larval brain is essential for the proper patterning and functionality of the adult central nervous system. Initiation of neuroblast proliferation requires signaling by the Fibroblast Growth Factor homolog Branchless and by the Hedgehog growth factor. We show here that the Branchless and Hedgehog pathways form a positive feedback loop to regulate the onset of neuroblast division. This feedback loop is initiated during embryogenesis. Our genetic and molecular studies demonstrate that the absolute level of Branchless and Hedgehog signaling is critical to fully activate stem cell division. Furthermore, over-expression and mutant studies establish that signaling by Branchless is the crucial output of the feedback loop that stimulates neuroblast division and that Branchless signaling is necessary for initiating the division of all mitotically regulated neuroblasts in the brain lobes. These studies establish the molecular mechanism through which Branchless and Hedgehog signaling interface to regulate the activation of neural stem cell division.

Mutation of the central nervous system neuroblast proliferation repressor ana leads to defects in larval olfactory behavior

In the developing nervous system, interactions between glia and immature neurons or neuroblasts regulate axon pathfinding, migration, and cell division, and therefore affect structure and function. Glial control of neuroblast cell division has been documented by studies of the anachronism (ana) gene of Drosophila melanogaster. ana encodes a glycoprotein which, in the developing larval central nervous system, is secreted by glia that neighbor regulated neuroblasts. Mutations in ana lead to premature neuroblast proliferation in the larval brain. Examination of lacZ expression from an ana enhancer trap line as well as detection of the ana protein show that ana is also expressed in the larval antennal-maxillary complex (AMC) at all larval stages. As previously reported for the central nervous system, ana expression in the AMC appears to be confined to glial cells. Larval olfactory system function in ana mutants was assayed in a behavioral paradigm. When tested with the three different chemoattractants, third instar ana9 mutant larvae showed diminished olfactory response compared to controls. Examination of a second ana allele revealed aberrant olfactory response to ethyl acetate, demonstrating that more than one mutation in ana can give rise to abnormal larval olfactory behavior. Assays of early first instar ana9 mutant larvae revealed defective olfactory behavior, implying that the olfactory phenotype stems from early larval AMC and/or embryonic origins. This is consistent with proliferation analysis in the early larval AMC region which uncovered a significantly higher number of S-phase cells in ana9 mutants.

Drosophila homeobox gene eve enhances trol, an activator of neuroblast proliferation in the larval CNS

The regulation of stem cell division by developmental cues is critical for the assembly and function of multicellular organisms. Stem cell division in the Drosophila brain is controlled by trol, which is required for activation of proliferation by quiescent neuroblasts at the appropriate stage of larval development. We show that the transcriptional regulator eve is part of the trol activation pathway by identifying eve as a dominant enhancer of a weak trol allele, trolb22. Known eve mutations are capable of enhancing the lethality of trolb22 and uncovering a defective neuroblast proliferation phenotype. Additionally, genetic and molecular analysis reveals that an independent mutation which acts as a dominant enhancer of trol is also an allele of eve. The enhancement of trolb22 lethality can be suppressed by the presence of an eve transgene. Interestingly, extra copies of eve supplied by the eve transgene also enhance trolb22 lethality, suggesting that the level of Eve protein may be critical for neuroblast activation. Finally, activation of neuroblast proliferation is normal in eve4 heterozygotes, suggesting that the proliferation defect observed in trolb22;eve/+ animals is due to a synergistic interaction.