Smooth muscle protein-22-mediated deletion of Tsc1 results in cardiac hypertrophy that is mTORC1-mediated and reversed by rapamycin.

Constitutive activation of mammalian target of rapamycin complex 1 (mTORC1), a key kinase complex that regulates cell size and growth, is observed with inactivating mutations of either of the tuberous sclerosis complex (TSC) genes, Tsc1 and Tsc2. Tsc1 and Tsc2 are highly expressed in cardiovascular tissue but their functional role there is unknown. We generated a tissue-specific knock-out of Tsc1, using a conditional allele of Tsc1 and a cre recombinase allele regulated by the smooth muscle protein-22 (SM22) promoter (Tsc1c/cSM22cre+/-) to constitutively activate mTOR in cardiovascular tissue. Significant gene recombination (∼80%) occurred in the heart by embryonic day (E) 15, and reduction in Tsc1 expression with increased levels of phosphorylated S6 kinase (S6K) and S6 was observed, consistent with constitutive activation of mTORC1. Cardiac hypertrophy was evident by E15 with post-natal progression to heart weights of 142 ± 24 mg in Tsc1c/cSM22cre+/- mice versus 65 ± 14 mg in controls (P < 0.01). Median survival of Tsc1c/cSM22cre+/- mice was 24 days, with none surviving beyond 6 weeks. Pathologic and echocardiographic analysis revealed severe biventricular hypertrophy without evidence of fibrosis or myocyte disarray, and significant reduction in the left ventricular end-diastolic diameter (P < 0.001) and fractional index (P < 0.001). Inhibition of mTORC1 by rapamycin resulted in prolonged survival of Tsc1c/cSM22cre+/- mice, with regression of ventricular hypertrophy. These data support a critical role for the Tsc1/Tsc2-mTORC1-S6K axis in the normal development of cardiovascular tissue and also suggest possible therapeutic potential of rapamycin in cardiac disorders where pathologic mTORC1 activation occurs.

Selective inhibition of growth of tuberous sclerosis complex 2 null cells by atorvastatin is associated with impaired Rheb and Rho GTPase function and reduced mTOR/S6 kinase activity.

Inactivating mutations in the tuberous sclerosis complex 2 (TSC2) gene, which encodes tuberin, result in the development of TSC and lymphangioleiomyomatosis (LAM). The tumor suppressor effect of tuberin lies in its GTPase-activating protein activity toward Ras homologue enriched in brain (Rheb), a Ras GTPase superfamily member. The statins, 3-hydroxy-3-methylglutaryl CoA reductase inhibitors, have pleiotropic effects which may involve interference with the isoprenylation of Ras and Rho GTPases. We show that atorvastatin selectively inhibits the proliferation of Tsc2-/- mouse embryo fibroblasts and ELT-3 smooth muscle cells in response to serum and estrogen, and under serum-free conditions. The isoprenoids farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP) significantly reverse atorvastatin-induced inhibition of Tsc2-/- cell growth, suggesting that atorvastatin dually targets a farnesylated protein, such as Rheb, and a geranylgeranylated protein, such as Rho, both of which have elevated activity in Tsc2-/- cells. Atorvastatin reduced Rheb isoprenylation, GTP loading, and membrane localization. Atorvastatin also inhibited the constitutive phosphorylation of mammalian target of rapamycin, S6 kinase, and S6 found in Tsc2-/- cells in an FPP-reversible manner and attenuated the high levels of phosphorylated S6 in Tsc2-heterozygous mice. Atorvastatin, but not rapamycin, attenuated the increased levels of activated RhoA in Tsc2-/- cells, and this was reversed by GGPP. These results suggest that atorvastatin may inhibit both rapamycin-sensitive and rapamycin-insensitive mechanisms of tuberin-null cell growth, likely via Rheb and Rho inhibition, respectively. Atorvastatin may have potential therapeutic benefit in TSC syndromes, including LAM.

Renal and liver tumors in Tsc2(+/-) mice, a model of tuberous sclerosis complex, do not respond to treatment with atorvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor.

Inactivating mutations of the tumor suppressor gene TSC2 are associated with tumorigenesis in tuberous sclerosis complex (TSC) and lymphangioleiomyomatosis. Statins, as 3-hydroxy-3-methylglutaryl CoA reductase inhibitors, have the potential to limit the growth of these tumors by limiting the isoprenylation of activated GTPases in Tsc2-null cells. We tested atorvastatin as a therapy for (a) ethylnitrosourea (ENU)-enhanced renal cystadenoma and (b) spontaneous liver hemangioma in 129Sv/Jae Tsc2(+/-) mice. ENU-treated Tsc2(+/-) mice were given atorvastatin chow (0.1%, w/w) for 1 or 3 months before sacrifice at 6 months; 129Sv/Jae Tsc2(+/-) mice were given atorvastatin chow (0.1%, w/w) for 6 months before sacrifice at 12 months. All treatment groups were compared with mice of identical genotype and strain background that were fed control chow. Pathologic analyses revealed a predominance of renal cystadenoma in ENU-treated and liver hemangioma in non-ENU-treated 129Sv/Jae Tsc2(+/-) mice. In both cohorts, serum cholesterol levels and levels of phosphorylated S6 and GTP-RhoA in healthy tissue were significantly (>50%) reduced in atorvastatin-treated mice as compared with controls. Following atorvastatin treatment, no significant reduction in tumor size, morphology, or phosphorylated S6 levels was observed for either ENU-associated renal cystadenoma or spontaneous liver hemangioma as compared with the untreated groups. In conclusion, although the marked reduction in cholesterol levels indicates that atorvastatin was effective as an 3-hydroxy-3-methylglutaryl CoA reductase inhibitor, it did not inhibit the growth of tumors that develop in these Tsc2(+/-) models, suggesting that it is unlikely to have benefit as a single-agent therapy for TSC-associated tumors.

Discovery of a small molecule that blocks wall teichoic acid biosynthesis in Staphylococcus aureus.

Both Gram-positive and Gram-negative bacteria contain bactoprenol-dependent biosynthetic pathways expressing non-essential cell surface polysaccharides that function as virulence factors. Although these polymers are not required for bacterial viability in vitro, genes in many of the biosynthetic pathways are conditionally essential: they cannot be deleted except in strains incapable of initiating polymer synthesis. We report a cell-based, pathway-specific strategy to screen for small molecule inhibitors of conditionally essential enzymes. The screen identifies molecules that prevent the growth of a wildtype bacterial strain but do not affect the growth of a mutant strain incapable of initiating polymer synthesis. We have applied this approach to discover inhibitors of wall teichoic acid (WTA) biosynthesis in Staphylococcus aureus. WTAs are anionic cell surface polysaccharides required for host colonization that have been suggested as targets for new antimicrobials. We have identified a small molecule, 7-chloro-N,N-diethyl-3-(phenylsulfonyl)-[1,2,3]triazolo[1,5-a]quinolin-5-amine (1835F03), that inhibits the growth of a panel of S. aureus strains (MIC = 1-3 microg mL(-1)), including clinical methicillin-resistant S. aureus (MRSA) isolates. Using a combination of biochemistry and genetics, we have identified the molecular target as TarG, the transmembrane component of the ABC transporter that exports WTAs to the cell surface. We also show that preventing the completion of WTA biosynthesis once it has been initiated triggers growth arrest. The discovery of 1835F03 validates our chemical genetics strategy for identifying inhibitors of conditionally essential enzymes, and the strategy should be applicable to many other bactoprenol-dependent biosynthetic pathways in the pursuit of novel antibacterials and probes of bacterial stress responses.

Low temperature (23 degrees C) increases expression of biofilm-, cold-shock- and RpoS-dependent genes in Escherichia coli K-12.

Temperature serves as a cue to regulate gene expression in Escherichia coli and other bacteria. Using DNA microarrays, we identified 297 genes whose expression is increased at 23 degrees C compared to 37 degrees C in E. coli K-12. Of these genes, 122 are RpoS-controlled, confirming genome-wide the model that low temperature serves as a primary cue to trigger the general stress response. Several genes expressed at 23 degrees C overlap with the cold-shock response, suggesting that strategies used to adapt to sudden shifts in temperature also mediate long-term growth at 23 degrees C. Another category of genes more highly expressed at 23 degrees C are associated with biofilm development, implicating temperature as an important cue influencing this developmental pathway. In a candidate set of genes tested, the biofilm genes (adrA, bolA, mlrA, nhaR, csgA, yceP/bssS) and cold-shock genes (otsA, yceP/bssS) were found to be RpoS- and DsrA-dependent for their transcription at 23 degrees C. In contrast, transcription of three genes (ycgZ, dps and ymgB) was either partially or fully independent of these regulators, signifying there is an alternative thermoregulatory mechanism(s) that increases gene expression at 23 degrees C. Increased expression at 23 degrees C compared to 37 degrees C is retained in various media tested for most of the genes, supporting the relative importance of this cue in adaptation to changing environments. Both the RpoS-dependent gene otsA and the RpoS-independent gene ymgB demonstrated increased expression levels within 1 h after a shift from 37 to 23 degrees C, indicating a rapid response to this environmental cue. Despite changes in gene expression for many RpoS-dependent genes, experiments assessing growth rate at 23 degrees C and viability at 4 degrees C did not demonstrate significant impairment in rpoS : : Tn10 or dsrA : : cat mutant strains in comparison to the wild-type strain. Biofilm formation was favoured at low temperature and is moderately impaired in both the rpoS : : Tn10 and dsrA : : cat mutants at 23 degrees C, suggesting genes controlled by these regulators play a role necessary for optimal biofilm formation at 23 degrees C. Taken together, our data demonstrate that a large number of genes are increased in expression at 23 degrees C to globally respond to this environmental change and that at least two thermoregulatory pathways are involved in co-ordinating this response - the RpoS/DsrA pathway and an alternative thermoregulatory pathway, independent of these regulators.

Human body temperature (37degrees C) increases the expression of iron, carbohydrate, and amino acid utilization genes in Escherichia coli K-12.

Using DNA microarrays, we identified 126 genes in Escherichia coli K-12 whose expression is increased at human body temperature (37 degrees C) compared to growth at 23 degrees C. Genes involved in the uptake and utilization of amino acids, carbohydrates, and iron dominated the list, supporting a model in which temperature serves as a host cue to increase expression of bacterial genes needed for growth. Using quantitative real-time PCR, we investigated the thermoregulatory response for representative genes in each of these three categories (hisJ, cysP, srlE, garP, fes, and cirA), along with the fimbrial gene papB. Increased expression at 37 degrees C compared to 23 degrees C was retained in both exponential and stationary phases for all of the genes and in most of the various media tested, supporting the relative importance of this cue in adapting to changing environments. Because iron acquisition is important for both growth and virulence, we analyzed the regulation of the iron utilization genes cirA and fes and found that growth in iron-depleted medium abrogated the thermoregulatory effect, with high-level expression at both temperatures, contrasting with papB thermoregulation, which was not greatly altered by limiting iron levels. A positive role for the environmental regulator H-NS was found for fes, cirA, hisJ, and srlE transcription, whereas it had a primarily negative effect on cysP and garP expression. Together, these studies indicate that temperature is a broadly used cue for regulating gene expression in E. coli and that H-NS regulates iron, carbohydrate, and amino acid utilization gene expression.