The activity of TGF-beta1 is regulated primarily extracellularly where the secreted latent form must be modified to expose the active molecule. Here we show that thrombospondin-1 is responsible for a significant proportion of the activation of TGF-beta1 in vivo. Histological abnormalities in young TGF-beta1 null and thrombospondin-1 null mice were strikingly similar in nine organ systems. Lung and pancreas pathologies similar to those observed in TGF-beta1 null animals could be induced in wild-type pups by systemic treatment with a peptide that blocked the activation of TGF-beta1 by thrombospondin-1. Although these organs produced little active TGF-beta1 in thrombospondin null mice, when pups were treated with a peptide derived from thrombospondin-1 that could activate TGF-beta1, active cytokine was detected in situ, and the lung and pancreatic abnormalities reverted toward wild type.

Angiogenesis sustains tumor growth and metastasis, and recent studies indicate that the vascular endothelium regulates tissue mass. In the prostate, androgens drive angiogenic inducers to stimulate growth, whereas androgen withdrawal leads to decreased vascular endothelial growth factor, vascular regression and epithelial cell apoptosis. Here, we identify the angiogenesis inhibitor pigment epithelium-derived factor (PEDF) as a key inhibitor of stromal vasculature and epithelial tissue growth in mouse prostate and pancreas. In PEDF-deficient mice, stromal vessels were increased and associated with epithelial cell hyperplasia. Androgens inhibited prostatic PEDF expression in cultured cells. In vivo, androgen ablation increased PEDF in normal rat prostates and in human cancer biopsies. Exogenous PEDF induced tumor epithelial apoptosis in vitro and limited in vivo tumor xenograft growth, triggering endothelial apoptosis. Thus, PEDF regulates normal pancreas and prostate mass. Its androgen sensitivity makes PEDF a likely contributor to the anticancer effects of androgen ablation.

No abstract

Aberrant blood vessel growth in the retina that underlies the pathology of proliferative diabetic retinopathy and retinopathy of prematurity is the result of the ischemia-driven disruption of the normally antiangiogenic environment of the retina. In this study, we show that a potent inhibitor of angiogenesis found naturally in the normal eye, pigment epithelium-derived growth factor (PEDF), inhibits such aberrant blood vessel growth in a murine model of ischemia-induced retinopathy. Inhibition was proportional to dose and systemic delivery of recombinant protein at daily doses as low as 2.2 mg͞kg could prevent aberrant endothelial cells from crossing the inner limiting membrane. PEDF appeared to inhibit angiogenesis by causing apoptosis of activated endothelial cells, because it induced apoptosis in cultured endothelial cells and an 8-fold increase in apoptotic endothelial cells could be detected in situ when the ischemic retinas of PEDF-treated animals were compared with vehicle-treated controls. The ability of low doses of PEDF to curtail aberrant growth of ocular endothelial cells without overt harm to retinal morphology suggests that this natural protein may be beneficial in the treatment of a variety of retinal vasculopathies.

Peroxisome proliferator activator receptor (PPAR)-binding protein (PBP) is an important coactivator forPPAR␥ and other nuclear receptors. It has been identified as an integral component of a multiprotein thyroid hormone receptor-associated protein/vitamin D 3 receptor-interacting protein/activator-recruited cofactor complexes required for transcriptional activity. Here, we show that PBP is critical for the development of placenta and for the normal embryonic development of the heart, eye, vascular, and hematopoietic systems. The primary functional cause of embryonic lethality at embryonic day11.5 observed with PBP null mutation was cardiac failure because of noncompaction of the ventricular myocardium and resultant ventricular dilatation. There was a paucity of retinal pigment, defective lens formation, excessive systemic angiogenesis, a deficiency in the number of megakaryocytes, and an arrest in erythrocytic differentiation. Some of these defects involve PPAR␥ and retinoid-sensitive sites, whereas others have not been recognized in the PPARsignaling pathway. Phenotypic changes in four organ systems observed in PBP null mice overlapped with those in mice deficient in members of GATA, a family of transcription factors known to regulate differentiation of megakaryocytes, erythrocytes, and adipocytes. We demonstrate that PBP interacts with all five GATA factors analyzed, GATA-1, GATA-2, GATA-3, GATA-4, and GATA-6, and show that the binding of GATA-1, GATA-4, and GATA-6 to PBP is not dependent on the nuclear receptor recognition sequence motif LXXLL (where L is leucine and X is any amino acid) in PBP. Coexpression of PBP with GATA-3 markedly enhanced transcriptional activity of GATA-3 in nonhematopoietic cells. These observations identify the GATA family of transcription factors as a new interacting partner of PBP and demonstrate that PBP is essential for normal development of vital organ systems.

Nuclear receptor coactivator PRIP (peroxisome proliferator-activated receptor (PPAR␥)-interacting protein) and PRIP-interacting protein with methyltransferase activity, designated PIMT, appear to serve as linkers between cAMP response element-binding protein-binding protein (CBP)/p300-anchored and PBP (PPAR␥-binding protein)-anchored coactivator complexes involved in the transcriptional activity of nuclear receptors. To assess the biological significance of PRIP, we disrupted the PRIP gene in mice by homologous recombination. Mice nullizygous for PRIP died between embryonic day 11.5 and 12.5 (postcoitum) due in most part to defects in the development of placenta, heart, liver, nervous system, and retardation of embryonic growth. Transient transfection assays using fibroblasts isolated from PRIP ؊/؊ embryos revealed a significant decrease in the capacity for ligand-dependent transcriptional activation of retinoid X receptor ␣ and to a lesser effect on PPAR␥ transcriptional activity. These observations indicate that PRIP like PBP, CBP, and p300 is an essential and nonredundant coactivator.Our understanding of the mechanisms underlying transcriptional activation by nuclear receptors has been advanced by the identification of nuclear receptor coactivators or coregulators that appear to influence embryonic development, cell proliferation, and differentiation (1). These include p160/SRC-1 1 (steroid receptor coactivator-1) family with three members (SRC-1, TIF/GRIP1/SRC-2, and pCIP/AIB1/ACTR/RAC3/TRAM1/SRC3) (2-6), CREB-binding protein (CBP) (7), adenovirus E1A-binding protein p300 (8), peroxisome proliferator-activated receptor-␥ (PPAR␥)-binding protein (PBP) (9), PPAR-interacting protein (PRIP/ASC-2/RAP250/TRBP/NRC) (10 -14) and PPAR␥ coactivator-1 (PGC-1) (15), among others. Nuclear receptor coactivators contain one or more conserved LXXLL (where L is leucine and X any amino acid) signature motif, which has been found to be necessary and sufficient for ligand-dependent interactions with the activation function-2 domain present in the C-terminal hormone-binding region of the nuclear receptors (1, 6). It is generally held that coactivators play a central role in mediating nuclear receptor transcriptional activity by functioning as at least two large multiprotein complexes formed either sequentially or combinatorially (1). The first complex anchored by CBP/p300 and containing p/160 cofactors/SRC-1 cofactors exhibits histone acetyltransferase activity necessary for remodeling chromatin (1,4,7,16), while the second multiprotein complex, variously referred to as TRAP/DRIP/ARC mediator complex, which is anchored by PBP (17-19), facilitates interaction with RNA polymerase II complexes of the basal transcription machinery (1). Deletion of CBP/p300 and PBP genes in the mouse results in embryonic lethality around E11.5 days, indicating that disruption of these pivotal anchoring coactivators affects the integrity of the cofactor complexes, thus altering the function of many nuclear receptors and most likely of other transcript...

Chronic bacterial infection and neutrophilic inflammation characterize cystic fibrosis (CF) pulmonary disease. In many disorders, inflammation and angiogenesis are codependent phenomena. We previously noted excessive angiogenesis in CF tissues and elevated vascular endothelial growth factor (VEGF) in random serum samples from subjects with CF. To further explore this finding, we measured serum VEGF in 38 subjects with stable CF and in 25 subjects with other pulmonary diseases. Mean VEGF was elevated in both groups compared with reference values, but it was higher in CF: 403 +/- 280 versus 255 +/- 169 pg/ml, p = 0.02. VEGF was negatively correlated with FEV(1) in CF, r = -0.51, p = 0.007. To assess the effect of airway infection on VEGF, 10 subjects with CF were studied before and after intravenous antibiotic therapy for pulmonary exacerbation. VEGF levels decreased with antibiotic therapy, from 537 +/- 220 to 259 +/- 176 pg/ml, p = 0.001. We conclude that circulating VEGF is increased in subjects with CF and other inflammatory pulmonary disorders. In CF, VEGF elevation is related to airway infection. We speculate that increased circulating VEGF is related to chronic inflammation, which is robust in CF. Elevated circulating VEGF may result in tissue angiogenesis, furthering the progression of pulmonary disease.

Vitamin A and other retinoids profoundly inhibit morphological and biochemical features of epidermal differentiation in vivo and in vitro. To elucidate the molecular mechanism underlying the differential expression of epidermal keratins and their regulation by retinoids, we examined retinoid-mediated changes in tota protein expression, protein synthesis, mRNA expression, and transcription in cultured human keratinocytes and in squamous cell carcinoma (SCC-13) cells of epidermal origin. Our studies revealed that the epidermal keratins, K5, K6, K14, and K16, their mRNAs, and their transcripts were diminished relative to actin as a consequence of retinoic acid (RA) treatment. The effects were most pronounced in SCC-13 and were detected as early as 6 hr post-RA treatment, with enhancement over an additional 24-48 hr. Repression was also observed when 5' upstream sequences of K14 or K5 genes were used to drive expression of a chloramphenicol acetyltransferase reporter gene in SCC-13 keratinocytes. Both cell types were found to express mRNAs for the