RB loss occurs commonly in neoplasia but its contributions to advanced cancer have not been assessed directly. Here we show that RB loss in multiple murine models of cancer produces a prometastatic phenotype. Gene expression analyses showed that regulation of the cell motility receptor RHAMM by the RB/E2F pathway was critical for epithelial–mesenchymal transition, motility, and invasion by cancer cells. Genetic modulation or pharmacologic inhibition of RHAMM activity was sufficient and necessary for metastatic phenotypes induced by RB loss in prostate cancer. Mechanistic studies in this setting established that RHAMM stabilized F-actin polymerization by controlling ROCK signaling. Collectively, our findings show how RB loss drives metastatic capacity and highlight RHAMM as a candidate therapeutic target for treating advanced prostate cancer.
A large number of mitochondrial proteins lack canonical mitochondrial-targeting signals. The bimodal transport of cytochromes P450 (CYPs) to endoplasmic reticulum and mitochondria (MT), reported previously by us, likely represents one mode of non-canonical protein targeting to MT. Herein, we have studied the mechanism of mouse MT-CYP1A1 targeting to gain insight into the regulatory features and evolutionary conservation of bimodal targeting mechanism. Mouse MT-CYP1A1 consists of two NH 2 -terminal-truncated molecular species, ؉91A1 and ؉331A1. Mutations Pro-2 3 Leu and Tyr-5 3 Leu, which increase the signal recognition particle ( Proteins targeted to mitochondria contain either NH 2 -terminal or internal signals, which include amphipathic ␣-helices, -sheet, and random structures with spaced positively charged residues (1-3). A majority of the NH 2 -terminally located mitochondrial-targeting signals are cleaved by the mitochondrial matrix metalloprotease following their import, although in a number of cases the signals remain uncleaved (1-4). Recent proteomic studies indicate that yeast mitochondria contain as many as 800 proteins while the rodent heart and liver mitochondria may contain well over 1500 proteins (5-8). It is estimated that Ͼ50% of mitochondria-associated proteins in both yeast and mammalian cells lack canonical MT 4 -targeting signals, and the precise mode of targeting of these proteins as well as mechanism of their translocation across the MT membranes remain unclear. The bimodal targeting of CYPs to ER and MT, Alzheimer amyloid precursor protein to plasma membrane and MT, and translocation of cytosolic Glutathione S-transferases to MT (9 -14) may represent one mode of targeting of noncanonical signal containing proteins to the MT compartment.Recent studies from our laboratory showed that different xenobiotic-inducible CYPs such as rat CYP1A1, CYP2E1, and CYP2B1 and others that are widely recognized as microsomal proteins (MC-CYPs) are also targeted to varying degrees to mitochondria (9 -11). These studies led to the concept of a new family of chimeric non-canonical-targeting signals, which function both as ER-targeting and MT-targeting signals, under different physiological conditions. We proposed that bimodal targeting of these CYP proteins is facilitated by the cryptic MTtargeting signal domain (residues 29 -40), located between the transmembrane helical domain (residues 1-30) and the Prorich domain (residues 39 -44) in various members of the CYP1, CYP2, and CYP3 families. We have also shown that the cryptic MT-targeting signals of different CYPs are activated by two distinct mechanisms: (a) endoproteolytic cleavage of the NH 2 -
The biochemical and molecular characteristics of cytochrome P4501A1 targeted to rat brain mitochondria was studied to determine the generality of the targeting mechanism previously described for mitochondrial cytochrome P450MT2 (P450MT2) from rat liver. In rat brain and C6 glioma cells chronically exposed to -naphoflavone (BNF), P450MT2 content reached 50 and 95% of the total cellular pool, respectively. P450MT2 from 10 days of BNF-treated rat brain was purified to over 85% purity using hydrophobic chromatography followed by adrenodoxin affinity binding. Purified brain P450MT2 consisted of two distinct molecular species with NH 2 termini identical to liver mitochondrial forms. These results confirm the specificity of endoprotease-processing sites. The purified P450MT2 showed a preference for adrenodoxin ؉ adrenodoxin reductase electron donor system and exhibited high erythromycin N-demethylation activity. Brain mitoplasts from 10-day BNF-treated rats and also purified P450MT2 exhibited high N-demethylation activities for a number of neuroactive drugs, including trycyclic anti-depressants, anti-convulsants, and opiates. At 10 days of BNF treatment, the mitochondrial metabolism of these neuroactive drugs represented about 85% of the total tissue activity. These results provide new insights on the role of P450MT2 in modulating the pharmacological potencies of different neuroactive drugs in chronically exposed individuals.Cytochrome P450 (P450) 1 enzymes play a critical role in the metabolism of an array of endogenous as well as exogenous substrates (1-3). The xenobiotic inducible forms with roles in the metabolism of carcinogens, pollutants, and drugs were thought to be exclusively associated with the endoplasmic reticulum (hereafter referred to as microsomes) of liver, brain, and other tissues. In contrast to this general belief, recent reports from our laboratory showed that the BNF-inducible P4501A1 and phenobarbital-inducible P4502B1 are also targeted to mitochondria under both in vitro and in vivo cell transfection conditions. These results are consistent with previous studies from our, as well as, other laboratories showing the presence of P450 proteins cross-reacting with antibodies to the major microsomal forms, in liver and brain mitochondria from inducer treated and untreated rats, and also insects (4 -10). Direct sequencing of hepatic mitochondrial P450 proteins purified from BNF-treated rats suggested the occurrence of two forms of P450MT2, both of which were NH 2 -terminal cleaved versions of P4501A1 (11). The molecular form cleaved past the 4th amino acid residue (ϩ5/1A1) represents a minor component while that cleaved past residue 32 (ϩ33/1A1) represents the major component in mitochondria from BNF-treated rat liver. This major form will be routinely referred to as P450MT2 throughout this paper.The mitochondrial targeted P450MT2 exhibited many molecular and biochemical properties distinct from the parent microsomal P4501A1 (12): 1) P450MT2 interacted with Adx with an affinity of 0.6 M K d , 2) funct...
SUMMARY Bimodal targeting of the endoplasmic reticular protein, cytochrome P4501A1 (CYP1A1), to mitochondria involves activation of a cryptic mitochondrial targeting signal through endoprotease processing of the protein. Here, we characterized the endoprotease regulating CYP1A1 mitochondrial targeting. The endoprotease, which was induced by β-naphthoflavone, was a dimer of 90 kDa and 40 kDa subunits, each containing Ser protease domains. The purified protease processed CYP1A1 in a sequence-specific manner, leading to its mitochondrial import. The glucocorticoid receptor, retinoid X receptor, and p53 underwent similar processing-coupled mitochondrial transport. The inducible 90 kDa subunit was a limiting factor in many cells and some tissues, and thus, regulates the mitochondrial levels of these proteins. A number of other mitochondria-associated proteins with non-canonical targeting signals may also be substrates of this endoprotease. Our findings point to a novel mechanism of mitochondrial protein import that requires an inducible cytosolic endoprotease for activation of cryptic mitochondrial targeting signals.
Hypertrophy occurs in urinary bladder wall smooth muscle (BSM) in men with partial bladder outlet obstruction (PBOO) caused by benign prostatic hyperplasia (BPH) and in animal models of PBOO. Hypertrophied BSM from the rabbit model exhibits downregulation of caveolin-1, a structural and functional protein of caveolae that function as signaling platforms to mediate interaction between receptor proteins and adaptor and effector molecules to regulate signal generation, amplification, and diversification. Caveolin-1 expression is diminished in PBOO-induced BSM hypertrophy in mice and in men with BPH. The proximal promoter of the human and mouse caveolin-1 (CAV1) gene was characterized, and it was observed that the transcription factor GATA-6 binds this promoter, causing reduced expression of caveolin-1. Furthermore, caveolin-1 expression levels inversely correlate with the abundance of GATA-6 in BSM hypertrophy in mice and human beings. Benign prostatic hyperplasia (BPH) is common in aging men and is often associated with lower urinary tract symptoms. Partial bladder outlet obstruction (PBOO) has long been considered a key factor in the mechanism through which BPH causes lower urinary tract symptoms. PBOO-induced lower urinary tract symptoms are associated with bladder wall smooth muscle (BSM) remodeling including hypertrophy, thereby altering bladder contractility.
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