Histone deacetylases (HDACs) represent an expanding family of protein modifying-enzymes that play important roles in cell proliferation, chromosome remodeling, and gene transcription. We have previously shown that recombinant human HDAC8 can be expressed in bacteria and retain its catalytic activity. To further explore the catalytic activity of HDACs, we expressed two additional human class I HDACs, HDAC1 and HDAC3, in baculovirus. Recombinant HDAC1 and HDAC3 fusion proteins remained soluble and catalytically active and were purified to near homogeneity. Interestingly, trichostatin (TSA) was found to be a potent inhibitor for all three HDACs (IC 50 value of ϳ0.1-0.3 M), whereas another HDAC inhibitor MS-27-275 (N-(2-aminophenyl)-4-[N-(pyridin-3-methyloxycarbonyl)-aminomethyl]benzamide) preferentially inhibited HDAC1 (IC 50 value of ϳ0.3 M) versus HDAC3 (IC 50 value of ϳ8 M) and had no inhibitory activity toward HDAC8 (IC 50 value Ͼ100 M). MS-27-275 as well as TSA increased histone H4 acetylation, induced apoptosis in the human colon cancer cell line SW620, and activated the simian virus 40 early promoter. HDAC1 protein was more abundantly expressed in SW620 cells compared with that of HDAC3 and HDAC8. Using purified recombinant HDAC proteins, we identified several novel HDAC inhibitors that preferentially inhibit HDAC1 or HDAC8. These inhibitors displayed distinct properties in inducing histone acetylation and reporter gene expression. These results suggest selective HDAC inhibitors could be identified using recombinantly expressed HDACs and that HDAC1 may be a promising therapeutic target for designing HDAC inhibitors for proliferative diseases such as cancer.
The mouse skin carcinogenesis protocol is a unique model for understanding the molecular events leading to oncogenic transformation. Mutations in the Ha-ras gene, and the presence of functional cyclin D1 and the EGF receptor, have proven to be important in this system. However, the signal transduction pathways connecting these elements during mouse skin carcinogenesis are poorly understood. This paper studies the relevance of the Akt and ERK pathways in the di erent stages of chemically induced mouse skin tumors. Akt activity increases throughout the entire process, and its early activation is detected prior to increased cyclin D1 expression. ERK activity rises only during the later stages of malignant conversion. The observed early increase in Akt activity appears to be due to raised PI-3K activity. Other factors acting on Akt such as ILK activation and decreased PTEN phosphatase activity appear to be involved at the conversion stage. To further con®rm the involvement of Akt in this process, PB keratinocytes were transfected with Akt and subsequently injected into nude mice. The expression of Akt accelerates tumorigenesis and contributes to increased malignancy of these keratinocytes as demonstrated by the rate of appearance, the growth and the histological characteristics of the tumors. Collectively, these data provide evidence that Akt activation is one of the key elements during the di erent steps of mouse skin tumorigenesis.
To elucidate the possible role of NF-kB in mouse skin carcinogenesis we studied the expression of p50 (NFkB1), p52 (NF-kB2), p65 (RelA) and IkB-a inhibitor as well as kB-binding activity in adult SENCAR mouse skin, skin papillomas, and squamous cell carcinomas (SCC) generated by a two-stage carcinogenesis protocol. We found that in normal epidermis all of the above proteins were mostly expressed in the cytoplasm of basal cells. Western blot analysis revealed a dramatic increase of p50 and p52 expression in mouse skin tumors starting from the middle stage of promotion. We also found that the level of IkB-a protein in many late papillomas and SCC was lower than in normal epidermis. Results of EMSA showed an increase in kB-binding activity in mouse skin tumors and suggested that p50 is the major component of constitutive kB-binding complexes in normal epidermis and in tumors. It has been shown that nuclear IkB protein Bcl-3 is able to increase p50/p50 homodimer binding to the di erent kB sites in mouse thymocytes. Our ®nding on Bcl-3 overexpression in late papillomas and SCC could explain the selective increase of p50-related kB-binding in mouse skin tumors. Thus, our results strongly suggest the important role of p50 in skin carcinogenesis.
Previous studies have indicated that Bcl-3 interacts through its ankyrin repeats with the transcriptional factors NF-B1 (p50) and NF-B2 (p52), affecting their biological activities. To further investigate the role of Bcl-3 in vivo and its association with the NF-B proteins, we have generated transgenic mice constitutively expressing Bcl-3 in thymocytes. The results indicate that Bcl-3 is associated with endogenous p50 and p52 in nuclear extracts from transgenic animals. Remarkably, constitutive expression of Bcl-3 in these cells augments the DNA binding activity of endogenous p50 homodimers more than 10-fold but does not significantly increase the activity of p52 homodimers. This effect could be reproduced in vitro and is blocked by anti-Bcl-3 antibodies. We have also shown that Bcl-3 is phosphorylated in thymocytes and that its dephosphorylation greatly decreases the effect on p50 homodimers.The bcl-3 gene was cloned from a recurrent chromosomal translocation, t(14;19)(q32;q13.1), present in human B-cell chronic lymphocytic leukemias (39,40,60,65). It is remarkable that all the rearrangements in this locus occur in the 5Ј regulatory region of the gene and result in increased expression of wild-type 45,65). This protein contains seven ankyrin repeats and shares structural features with I B␣, I B, I B␥, and the C terminus of NF-B2 (5, 24, 57).Members of the Rel/NF-B family of transcription factors play a major role as early mediators of immune and inflammatory responses (4, 37, 54). Members of this family are differentially expressed during mouse embryonic development and in adult lymphoid tissues (13,14,62). Each of these proteins contains a highly conserved region of approximately 300 amino acids called the Rel homology domain which is responsible for both DNA binding and dimerization. In mammalian cells, this protein family can be divided into two classes. One class includes the precursors NF-B1 (p105/p50) and NF-B2 (p100/p52), which are synthesized as inactive molecules and remain in the cytoplasm. These precursors (p105 and p100), upon proteolytic processing, will generate the DNA-binding subunits p50 and p52, respectively. The other class of proteins comprises RelA (p65), c-Rel, and RelB. These proteins do not undergo proteolytic processing, and they harbor transcriptional activation domains (36). The biological functions of p50, RelA, RelB, and c-Rel have recently been addressed by the generation of null mice (7,11,30,53,63).The primary Rel/NF-B transactivating complexes in cells appear to be homo-and heterodimers involving members of each subclass that bind to specific B sites and differ in their transcriptional activities. In unstimulated cells the Rel/NF-B dimers are associated with the inhibitor I B proteins and remain as an inactive pool in the cytoplasm. Upon stimulation by different agents, I B molecules are rapidly phosphorylated and degraded, allowing the NF-B dimers to translocate into the nucleus and regulate transcription through binding to the B sites (2,5,10,18,19,24,36,41,46,(56)(57)(58)(59...
The glucocorticoid (GC) receptor (GR) mediates the effects of physiological and pharmacological GC ligands and has a major role in cutaneous pathophysiology. To dissect the epithelial versus mesenchymal contribution of GR in developing and adult skin, we generated mice with keratinocyte-restricted GR inactivation (GR epidermal knockout or GR(EKO) mice). Developing and early postnatal GR(EKO) mice exhibited impaired epidermal barrier formation, abnormal keratinocyte differentiation, hyperproliferation, and stratum corneum (SC) fragility. At birth, GR(EKO) epidermis showed altered levels of epidermal differentiation complex genes, proteases and protease inhibitors which participate in SC maintenance, and innate immunity genes. Many upregulated genes, including S100a8/a9 and Tslp, also have increased expression in inflammatory skin diseases. Infiltration of macrophages and degranulating mast cells were observed in newborn GR(EKO) skin, hallmarks of atopic dermatitis. In addition to increased extracellular signal-regulated kinase activation, GR(EKO) newborn and adult epidermis had increased levels of phosphorylated signal transducer and activator of transcription 3, a feature of psoriasis. Although adult GR(EKO) epidermis had a mild phenotype of increased proliferation, perturbation of skin homeostasis with detergent or phorbol ester triggered an exaggerated proliferative and hyperkeratotic response relative to wild type. Together, our results show that epidermal loss of GR provokes skin barrier defects and cutaneous inflammation.
Glucocorticoids (GCs) regulate skin homeostasis and combat cutaneous inflammatory diseases; however, adverse effects of chronic GC treatments limit their therapeutic use. GCs bind and activate the GC receptor and the mineralocorticoid receptor (MR), transcription factors that recognize identical hormone responsive elements. Whether epidermal MR mediates beneficial or deleterious GC effects is of great interest for improving GC-based skin therapies. MR epidermal knockout mice exhibited increased keratinocyte proliferation and differentiation and showed resistance to GC-induced epidermal thinning. However, crucially, loss of epidermal MR rendered mice more sensitive to inflammatory stimuli and skin damage. MR epidermal knockout mice showed increased susceptibility to phorbol 12-myristate 13-acetate-induced inflammation with higher cytokine induction. Likewise, cultured MR epidermal knockout keratinocytes had increased phorbol 12-myristate 13-acetate-induced NF-κB activation, highlighting an anti-inflammatory function for MR. GC-induced transcription was reduced in MR epidermal knockout keratinocytes, at least partially due to decreased recruitment of GC receptor to hormone responsive element-containing sequences. Our results support a role for epidermal MR in adult skin homeostasis and demonstrate nonredundant roles for MR and GC receptor in mediating GC actions.
Glucocorticoids (GCs) are potent inhibitors of epidermal proliferation and effective anti-inflammatory compounds, which make them the drug of choice for a wide range of inflammatory and hyperproliferative skin disorders. GC action is mediated via the glucocorticoid receptor (GR). To study the role of GR in skin development and the molecular mechanisms underlying its action, we generated transgenic mice overexpressing GR in epidermis and other stratified epithelia, under the control of the keratin K5 promoter. Newborn mice show altered skin development, manifested as variable-sized skin lesions that range from epidermal hypoplasia and underdeveloped dysplastic hair follicles to a complete absence of this tissue. In the most affected individuals, skin was absent at the cranial and umbilical regions, and the vibrissae and eyebrows appear scarce, short, and curly. In addition, as a consequence of transgene expression in other ectodermally derived epithelia, K5-GR mice exhibited further abnormalities that strikingly resemble the clinical findings in patients with ectodermal dysplasia, which includes aplasia cutis congenita. In adult transgenic skin, topical application of the tumor promoter TPA did not elicit hyperplasia or transcriptional induction of several proinflammatory cytokines. This anti-inflammatory role of GR was due at least in part to interference with NF-kB, leading to a strong reduction in the kB-binding activity without altering the transcriptional levels of the inhibitor IkBa.
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