The inhibition of the expression of the small Rho GTPase Rac1 induces differentiation with no effect on cell proliferation in growing human adult keratinocytes.
; ; et al
in Journal of Cellular Biochemistry (2008), 103(3), 857-64
Rac1 is a Rho subfamily small GTPase which is highly expressed in epidermal keratinocytes. In mice the significance of Rac1 for the maintenance of the epidermis has been controversial. In keratinocytes ... [more ▼]
Rac1 is a Rho subfamily small GTPase which is highly expressed in epidermal keratinocytes. In mice the significance of Rac1 for the maintenance of the epidermis has been controversial. In keratinocytes from human origin, the role of Rac1 in the control of growth/differentiation is still obscure. In this study we used RNA interference to induce specific inhibition of Rac1 expression in cultured human keratinocytes and analyzed the consequences on proliferation and differentiation. We found that the autocrine proliferation of keratinocytes is unaltered by Rac1 silencing. However, the suppression of Rac1 induced premature differentiation as revealed by the expression of markers (keratin 10, involucrin), but the involved mechanism is independent of the activity of p38 mitogen-activated protein kinase. Rather, we found that the effects of Rac1 silencing on keratinocytes differentiation are concomitant with negative regulation of the Ser62/Thr58 phosphorylation on the transcription factor c-myc, a mechanism known to control post-translational stability of the c-myc protein. Thus, in growing human keratinocytes, Rac1 could impede the expression of premature differentiation markers, probably by exerting positive control on c-myc activity and its binding to specific promoters. [less ▲]Detailed reference viewed: 25 (4 ULg)
Modular changes of cis-regulatory elements from two functional Pit1 genes in the duplicated genome of Cyprinus carpio.
; ; et al
in Journal of Cellular Biochemistry (2006), 99(3), 905-21
The pituitary-specific transcription factor Pit1 is involved in its own regulation and in a network of transcriptional regulation of hypothalamo-hypophyseal factors including prolactin (PRL) and growth ... [more ▼]
The pituitary-specific transcription factor Pit1 is involved in its own regulation and in a network of transcriptional regulation of hypothalamo-hypophyseal factors including prolactin (PRL) and growth hormone (GH). In the ectotherm teleost Cyprinus carpio, Pit1 plays an important role in regulation of the adaptive response to seasonal environmental changes. Two Pit1 genes exist in carp, a tetraploid vertebrate and transcripts of both genes were detected by RT-PCR analysis. Powerful comparative analyses of the 5'-flanking regions revealed copy specific changes comprising modular functional units in the naturally evolved promoters. These include the precise replacement of four nucleotides around the transcription start site embedded in completely conserved regions extending upstream of the TATA-box, an additional transcription factor binding site in the 5'-UTR of gene-I and, instead, duplication of a 9 bp element in gene-II. Binding of nuclear factors was assessed by electro mobility shift assays using extracts from rat pituitary cells and carp pituitary. Binding was confirmed at one conserved Pit1, one conserved CREB and one consensus MTF1. Interestingly, two functional Pit1 sites and one putative MTF1 binding site are unique to the Pit1 gene-I. In situ hybridization experiments revealed that the expression of gene-I in winter carp was significantly stronger than that of gene-II. Our data suggest that the specific control elements identified in the proximal regulatory region are physiologically relevant for the function of the duplicated Pit1 genes in carp and highlight modular changes in the architecture of two Pit1 genes that evolved for at least 12 MYA in the same organism. [less ▲]Detailed reference viewed: 3 (2 ULg)
Transcription factor Pit-1 expression is modulated upon seasonal acclimatization of eurythermal ectotherms: Identification of two Pit-1 genes in the carp
; ; et al
in Journal of Cellular Biochemistry (1999), 75(4), 598-609
A second Pit-1 gene in carp (Cyprinus carpio), including the complete structural gene and 1.1 kb of promoter region, was identified and completely sequenced. The exon-intron structure was determined, and ... [more ▼]
A second Pit-1 gene in carp (Cyprinus carpio), including the complete structural gene and 1.1 kb of promoter region, was identified and completely sequenced. The exon-intron structure was determined, and reverse transcription-polymerase chain reaction (RT-PCR) experiments suggest that only one Pit-1 splice variant is present in carp pituitary. The effect of seasonal acclimatization on the extent of Pit-1 gene expression was studied in summer- and winter-acclimatized carp. Quantitative RT-PCR analysis revealed a clear increase of Pit-1 mRNA in the pituitaries from summer-acclimatized carp compared with the winter-adapted fish. In situ hybridization of pituitary gland sections with riboprobes representing the complete 5-transactivating region of carp Pit-1 depicted a significantly higher Pit-1 mRNA level in the rostral pars distalis of the summer-acclimatized fish where prolactin is expressed in a manner that resembles the seasonal increase observed in the proximal pars distalis and the pars intermedia. The cell- and temporal-specific transcription of Pit-1 supports its role in the molecular mechanisms that underly the acclimatization process undergone by eurythermal fish as a result of the physical effects of seasonal changes on their habitat. J. Cell. Biochem. 75:598-609, 1999. © 1999 Wiley-Liss, Inc. [less ▲]Detailed reference viewed: 14 (4 ULg)
Molecular Cloning of a Mutated Hoxb7 Cdna Encoding a Truncated Transactivating Homeodomain-Containing Protein
Chariot, Alain ; ; et al
in Journal of Cellular Biochemistry (1998), 71(1), 46-54
Homeodomain-containing proteins regulate, as transcription factors, the coordinated expression of genes involved in development, differentiation, and malignant transformation. We report here the molecular ... [more ▼]
Homeodomain-containing proteins regulate, as transcription factors, the coordinated expression of genes involved in development, differentiation, and malignant transformation. We report here the molecular cloning of a mutated HOXB7 transcript encoding a truncated homeodomain-containing protein in MCF7 cells. This is a new example of mutation affecting the coding region of a HOX gene. In addition, we detected two HOXB7 transcripts in several breast cell lines and demonstrated that both normal and mutated alleles were expressed at the RNA level in MCF7 cells as well as in a variety of breast tissues and lymphocytes, suggesting that a truncated HOXB7 protein might be expressed in vivo. Using transient co-transfection experiments, we demonstrated that both HOXB7 proteins can activate transcription from a consensus HOX binding sequence in breast cancer cells. Our results provide evidence that HOXB7 protein has transcription factor activity in vivo and that the two last amino acids do not contribute to this property. [less ▲]Detailed reference viewed: 16 (4 ULg)
Formation of the 67-kDa laminin receptor by acylation of the precursor.
; ; et al
in Journal of Cellular Biochemistry (1998), 69(3), 244-51
Even though the involvement of the 67-kDa laminin receptor (67LR) in tumor invasiveness has been clearly demonstrated, its molecular structure remains an open problem, since only a full-length gene ... [more ▼]
Even though the involvement of the 67-kDa laminin receptor (67LR) in tumor invasiveness has been clearly demonstrated, its molecular structure remains an open problem, since only a full-length gene encoding a 37-kDa precursor protein (37LRP) has been isolated so far. A pool of recently obtained monoclonal antibodies directed against the recombinant 37LRP molecule was used to investigate the processing that leads to the formation of the 67-kDa molecule. In soluble extracts of A431 human carcinoma cells, these reagents recognize the precursor molecule as well as the mature 67LR and a 120-kDa molecule. The recovery of these proteins was found to be strikingly dependent upon the cell solubilization conditions: the 67LR is soluble in NP-40-lysis buffer whereas the 37LRP is NP-40-insoluble. Inhibition of 67LR formation by cerulenin indicates that acylation is involved in the processing of the receptor. It is likely a palmitoylation process, as indicated by sensitivity of NP-40-soluble extracts to hydroxylamine treatment. Immunoblotting assays performed with a polyclonal serum directed against galectin3 showed that both the 67- and the 120-kDa proteins carry galectin3 epitopes whereas the 37LRP does not. These data suggest that the 67LR is a heterodimer stabilized by strong intramolecular hydrophobic interactions, carried by fatty acids bound to the 37LRP and to a galectin3 cross-reacting molecule. [less ▲]Detailed reference viewed: 16 (0 ULg)
New insights into the metastasis-associated 67 kD laminin receptor.
; Castronovo, Vincenzo ; et al
in Journal of Cellular Biochemistry (1997), 67(2), 155-65
The interactions between tumor cells and laminin or other components of the extracellular matrix have been shown to play an important role in tumor invasion and metastasis. These interactions are mediated ... [more ▼]
The interactions between tumor cells and laminin or other components of the extracellular matrix have been shown to play an important role in tumor invasion and metastasis. These interactions are mediated by different cell surface molecules, including the monomeric 67 kD laminin receptor. This molecule appears to be very peculiar since so, far only a full-length gene encoding a 37 kD precursor protein has been isolated and the mechanism by which the precursor reaches the mature form is not understood. Based on clinical data, which clearly demonstrate the importance of the receptor in tumor progression, studies were conducted to define the structure, expression, and function of this laminin receptor as a step toward developing therapeutic strategies that target this molecule. The data suggest that acylation of the precursor is the key mechanism in maturation of the 67 kD form. The function of the membrane receptor is to stabilize the binding of laminin to cell surface integrins, acting as an integrin-accessory molecule, although homology of the gene encoding the receptor precursor with other genes suggests additional functions. Downregulation of the receptor expression on tumor cells might open new therapeutic approaches to decrease tumor aggressiveness. [less ▲]Detailed reference viewed: 3 (0 ULg)
Heparin-binding domain, type 1 and type 2 repeats of thrombospondin mediate its interaction with human breast cancer cells.
; ; Cataldo, Didier et al
in Journal of Cellular Biochemistry (1996), 62(4), 431-42
Thrombospondin is an adhesive glycoprotein that promotes breast cancer cell adhesion to human vascular endothelial cells (Incardona et al., 1995). In this study, we have identified the molecular domains ... [more ▼]
Thrombospondin is an adhesive glycoprotein that promotes breast cancer cell adhesion to human vascular endothelial cells (Incardona et al., 1995). In this study, we have identified the molecular domains of thrombospondin that mediate its binding to specific receptors on the human breast adenocarcinoma cell line, MDA-MB-231. Two recombinant fragments from the amino-terminus (TSPN18 and TSPN28), and the fusion proteins of the type 1 and type 2 repeats of human thrombospondin, inhibited binding of radiolabeled thrombospondin to MDA-MB-231 cells in suspension by 40-60% at 50 micrograms/ml whereas the type 3 repeat, carboxy-terminus and unfused glutathione-S-transferase as well as the synthetic peptide Gly-Arg-Gly-Asp-Ser (500 micrograms/ml) had little or no effect. Heparin and various glycosaminoglycans as heparan sulfate, chondroitin sulfates A, B or C, and fucoidan inhibited thrombospondin binding to MDA-MB-231 cells by more than 60% whereas dextran sulfate had only little effect. Treatment of cells with heparitinase, chondroitinase ABC, and hyaluronidase, but not with neuraminidase, induced 30-50% inhibition of thrombospondin binding suggesting the participation of both heparan sulfate and chondroitin sulfate cell surface-associated molecules. Inhibition of proteoglycan sulfation by chlorate or inhibition of glycosaminoglycan chain formation by two beta-D-xylosides also led to a substantial inhibition of thrombospondin binding. Our results indicate that several domains within the thrombospondin molecule, namely the amino-terminus, type 1 and type 2 repeats, participate in its binding to specific receptors bearing sulfated glycosaminoglycans on MDA-MB-231 cells. Biological assays have indicated that, in addition to these domains, the peptide Gly-Arg-Gly-Asp-Ser inhibited MDA-MB-231 cell attachment to thrombospondin suggesting that the last type 3 repeat of the molecule may also contribute to its cell adhesive activity. [less ▲]Detailed reference viewed: 25 (0 ULg)
Shedding of the 67-kD laminin receptor by human cancer cells.
; ; Castronovo, Vincenzo et al
in Journal of Cellular Biochemistry (1996), 60(2), 226-34
The 67-kD laminin receptor (67LR) is a cell membrane-associated molecule exhibiting high affinity for the basement membrane glycoprotein, laminin. While export of the 67LR toward the extracellular matrix ... [more ▼]
The 67-kD laminin receptor (67LR) is a cell membrane-associated molecule exhibiting high affinity for the basement membrane glycoprotein, laminin. While export of the 67LR toward the extracellular matrix has been recently suggested by electron microscopy studies, there is to date no evidence of shedding of the 67LR from cells. Using two monoclonal antibodies directed against the 67LR, we developed a double-determinant radioimmunoassay that demonstrates that the 67LR is released from cancer cells into the culture medium. The shed molecule exhibited the same apparent molecular weight as that of the membrane-associated 67LR, suggesting that no proteolytic cleavage is involved in the process. Furthermore, we demonstrate that the 67LR is not anchored to the membrane through a glycolsyl-phosphatidylinositol bridge. However, the observation that lactose increased the release of 67LR suggests that a lectin-type interaction is involved in the cell membrane association of this laminin binding protein and the cell surface. Interestingly, the released 67LR recovered after HPLC gel filtration was found free as well as associated to high molecular weight complexes. The free 67LR retained its ability to bind to the cell surface. Our study is the first demonstration that the 67LR is effectively shed by cancer cells. The released free 67LR could play an important role in modulating interactions between cancer cells and laminin during tumor invasion and metastasis. [less ▲]Detailed reference viewed: 7 (0 ULg)
A complex composed of at least two HeLa nuclear proteins protects preferentially one DNA strand of the simple (gt)n(ga)m containing region of intron 2 in HLA-DRB genes.
; ; Muller, Marc et al
in Journal of Cellular Biochemistry (1994), 56(1), 74-85
Electrophoretic mobility shift assays reveal that HeLa nuclear proteins bind fast and with measurable affinity to target DNAs containing mixed simple repetitive (gt)n(ga)m stretches. Preincubation of the ... [more ▼]
Electrophoretic mobility shift assays reveal that HeLa nuclear proteins bind fast and with measurable affinity to target DNAs containing mixed simple repetitive (gt)n(ga)m stretches. Preincubation of the proteins at elevated temperature prevents the formation of the major DNA/protein complex in favour of several distinct assemblies. A similar pattern of retarded bands was observed employing higher salt concentrations in the binding reaction. Thus conformational changes of different proteins appear to influence the complex rather than alternating DNA structures. Separation of the total nuclear extract into a water soluble and an insoluble protein fraction leads to a complete loss of target DNA binding capability of the fractions. The binding capacity is restored by combining the two fractions suggesting that at least two protein components are necessary to form a complex with the target sequence. The proteins can be differentiated into heat sensitive, water soluble and temperature stable, water insoluble, respectively. Furthermore, specifically binding polypeptides are not detectable by Southwestern analyses, probably because the essential components are separated during electrophoresis. DNase I footprint analyses yield four different protein binding regions only on the (gt)n(ga)m harbouring strand. The footprints cover larger portions of the mixed simple repeat in addition to a portion 5' of the (gt)n part. Hence at least two nuclear protein components of unknown biological function have to be present simultaneously to protect preferentially the (gt)n(ga)m-containing strand of intron 2 in HLA-DRB genes. [less ▲]Detailed reference viewed: 19 (1 ULg)
Increase in epidermal growth factor receptor and its mRNA levels by parathyroid hormone (1-34) and parathyroid hormone-related protein (1-34) during differentiation of human trophoblast cells in culture
; ; Scippo, Marie-Louise et al
in Journal of Cellular Biochemistry (1993), 53(1), 32-42Detailed reference viewed: 4 (0 ULg)
Modulation of Collagen and Fibronectin Synthesis in Fibroblasts by Normal and Malignant Cells
Noël, Agnès ; Munaut, Carine ; et al
in Journal of Cellular Biochemistry (1992), 48(2), 150-61
The influence of various normal and malignant human cells on the level of collagen synthesis by human fibroblasts was tested in coculture. As revealed by immunoperoxidase staining, in cocultures with ... [more ▼]
The influence of various normal and malignant human cells on the level of collagen synthesis by human fibroblasts was tested in coculture. As revealed by immunoperoxidase staining, in cocultures with breast adenocarcinoma cells (MCF7, SA52, T47D) fibroblasts synthesized collagen while tumor cells did not. Fibroblasts displayed increased collagen production without change in the overall protein synthesis. Several other types of cells derived from normal human tissues (keratinocytes, normal mammary cells) or from fibrosarcoma, melanoma, cervical carcinoma, choriocarcinoma, or other breast adenocarcinoma (SW613, MDA, BT20) did not affect collagen synthesis of fibroblasts. Although to a lesser extent, this stimulating effect was reproduced by using the conditioned medium (CM) of the active cells but not with CM of the other cell types. A slight stimulation was also obtained when tumoral MCF7 cells and fibroblasts shared the same medium but were physically separated, suggesting that close contact was required for optimal stimulation of collagen synthesis. The collagen synthesis stimulating activity was not related to a modification of fibroblast proliferation rate. The production of collagen types I, III, and VI and fibronectin were increased in cocultures of fibroblasts with MCF7 cells. The increased synthesis of collagen types I and III and fibronectin was paralleled by similar changes in the steady-state level of their mRNAs. On the contrary, the increased production of collagen type VI appeared regulated at a post-transcriptional level. [less ▲]Detailed reference viewed: 21 (2 ULg)
In vitro and in vivo models of varicella-zoster virus persistence in the nervous system
Sadzot-Delvaux, Catherine ; Merville, Marie-Paule ; et al
in Journal of Cellular Biochemistry (1988), 12CDetailed reference viewed: 11 (3 ULg)