Biomedical subjects

H Drissi

Publications and source records attributed to H Drissi.

9 recordsLinked to original sources

PubMed2005

TNF-alpha expression is transcriptionally regulated by RANK ligand.

Tumor necrosis factor (TNF)-alpha is known for its osteoclastogenic and resorptive activities. Induction of osteoclastogenesis by receptor activator of NF-kappaB ligand (RANKL) is accompanied by increased TNF-alpha expression. In the present study we investigated the mechanism by which RANKL induces expression of TNF-alpha in osteoclast precursors. The macrophage-like cell-line, RAW 264.7 was used as a model for osteoclast precursors. To examine if RANKL-mediated increase in TNF-alpha expression involves increased stability of its transcript, RAW264.7 cells were treated with or without RANKL, and then a transcription inhibitor was added. At different time points, TNF-alpha and L32 mRNA levels were examined. TNF-alpha mRNA stability was not altered by RANKL. We next measured directly the transcription rate of TNF-alpha by a run-on assay and found that RANKL increases TNF-alpha transcription rate by 2.9-fold in RAW264.7 cells. We further characterized this transcriptional induction of TNF-alpha by RANKL. Gel shift assays using nuclear extracts derived from RANKL-treated RAW264.7 cells show increased specific NF-kappaB binding activity on the murine TNF-alpha promoter. Gliotoxin, known for its ability to inhibit NF-kappaB activation blocked RANKL-induced TNF-alpha expression. We finally used 1,260 bp of the murine TNF-alpha promoter fused to luciferase, as well as four mutants of this promoter carrying mutations in each of the four NF-kappaB sites to stably transfect RAW 264.7 cells. Reporter activity was increased in response to RANKL in wild type promoter transfected cells, whereas treatment of the mutants' transfected cells did not elicit reporter activity. In conclusion, RANKL induces TNF-alpha expression via a transcriptional mechanism, depending on the NF-kappaB sites in the TNF promoter.

Animals↗

PubMed2001

Smad-Runx interactions during chondrocyte maturation.

BACKGROUND: Intracellular signaling triggered by bone morphogenetic proteins (BMPs) results in activated Smad complexes that regulate transcription of BMP-responsive genes. However, the low specificity of Smad binding to regulatory sequences implies that additional tissue-specific transcription factors are also needed. Runx2 (Cbfal) is a transcription factor required for bone formation. We have examined the role of Smads and Runx2 in BMP induction of type X collagen, which is a marker of chondrocyte hypertrophy leading to endochondral bone formation. METHODS: Pre-hypertrophic chondrocytes from the cephalic portion of the chick embryo sternum were placed in culture in the presence or absence of rhBMP-2. Cultures were transiently transfected with DNA containing the BMP-responsive type X collagen promoter upstream of the luciferase gene. The cultures were also transfected with plasmids, causing over-expression of Smads or Runx2, or both. After 24-48 hours, cell extracts were examined for levels of luciferase expression. RESULTS: In the presence of BMP-2, chondrocytes over-expressing BMP-activated Smadl or Smad5 showed significant enhancement of luciferase production compared with that seen with BMP alone. This enhancement was not observed with over-expression of Smad2, a transforming growth factor beta (TGF-beta)-activated Smad. Overexpression of Runx2 in BMP-treated cultures increased transcriptional activity to levels similar to those seen with Smads 1 or 5. When chondrocytes were simultaneously transfected with both Runx2 and Smad 1 or 5, promoter activity was further increased, indicating that BMP-stimulated Smad activity can be augmented by increasing the levels of Runx2. CONCLUSIONS: These results implicate the skeletal tissue transcription factor Runx2 in regulation of chondrocyte hypertrophy and suggest that maximal transcription of the type X collagen gene in pre-hypertrophic chondrocytes involves interaction of BMP-stimulated Smads with Runx2. CLINICAL RELEVANCE: Many skeletal abnormalities are associated with impaired regulation of chondrocyte hypertrophy in growth plates. These studies demonstrate that both BMP-activated Smads and Runx2 levels can modulate chondrocyte transition to hypertrophy.

Animals↗

PubMed2000

Transcriptional autoregulation of the bone related CBFA1/RUNX2 gene.

The runt related transcription factor CBFA1 (AML3/PEBP2alphaA/RUNX2) regulates expression of several bone- and cartilage-related genes and is required for bone formation in vivo. The gene regulatory mechanisms that control activation and repression of CBFA1 gene transcription during osteoblast differentiation and skeletal development are essential for proper execution of the osteogenic program. We have therefore defined functional contributions of 5' regulatory sequences conserved in rat, mouse and human CBFA1 genes to transcription. Deletion analysis reveals that 0.6 kB of the bone-related rat or mouse CBFA1 promoter (P1, MASNS protein isoform) is sufficient to confer transcriptional activation, and that there are multiple promoter domains which positively and negatively regulate transcription. Progressive deletion of promoter segments between nt -351 and -92 causes a striking 30- to 100-fold combined decrease in promoter activity. Additionally, 5' UTR sequences repress reporter gene transcription 2- to 3-fold. Our data demonstrate that CBFA1 is a principal DNA binding protein interacting with the 5' region of the CBFA1 gene in osseous cells, that there are at least three CBFA1 recognition motifs in the rat CBFA1 promoter, and that there are three tandemly repeated CBFA1 sites within the 5' UTR. We find that forced expression of CBFA1 protein downregulates CBFA1 promoter activity and that a single CBFA1 site is sufficient for transcriptional autosuppression. Thus, our data indicate that the CBFA1 gene is autoregulated in part by negative feedback on its own promoter to stringently control CBFA1 gene expression and function during bone formation.

5' Untranslated Regions↗

PubMed1999

Skeletal unloading induces biphasic changes in insulin-like growth factor-I mRNA levels and osteoblast activity.

To determine the local mechanisms involved in the effects of skeletal unloading on bone formation, we studied the temporal pattern of mRNA levels for insulin-like growth factor-I (IGF-I), IGF-I receptor type I (IGF-IR), and transforming growth factor beta receptor type II (TGF-betaRII) in relation to osteoblast phenotypic markers and osteoblast activity in hindlimb suspended rats. Skeletal unloading decreased bone volume and the mineralizing and osteoblastic surfaces at 4, 7, and 14 days in the tibial metaphysis, whereas the mineral appositional rate returned to normal at 14 days of suspension. RT-PCR analysis showed that skeletal unloading decreased type 1 collagen (Col 1) and osteocalcin (OC) mRNA levels in metaphyseal bone at days 4 and 7, and the levels returned to normal at 14 days of suspension. Unloading also decreased mRNA levels for IGF-I, IGF-IR, and TGF-betaRII at 4-7 days in the metaphyseal bone. However, IGF-I and IGF-IR levels rose above normal at 14 days of suspension. The biphasic changes in IGF-I mRNA levels were strongly correlated with Col 1 and OC mRNA levels. The associated biphasic pattern of IGF-I/IGF-IR expression, osteoblast markers, and osteoblast activity strongly suggests an important role for IGF-I signaling in the local effect of skeletal unloading on metaphyseal bone formation.

Animals↗

PubMed1999

The cell cycle regulator p27kip1 contributes to growth and differentiation of osteoblasts.

The cyclin-dependent kinase (cdk) inhibitors are key regulators of cell cycle progression. p27 and p21 are members of the Cip/Kip family of cdk inhibitors and regulate cell growth by inactivating cell cycle stage-specific CDK-cyclin complexes. Because down-regulation of osteoprogenitor proliferation is a critical step for osteoblast differentiation, we investigated expression of p27 and p21 during development of the osteoblast phenotype in rat calvarial osteoblasts and in proliferating and growth-inhibited osteosarcoma ROS 17/2.8 cells. Expression of these proteins indicates that p21, which predominates in the growth period, is related to proliferation control. p27 levels are maximal postproliferatively, suggesting a role in the transition from cell proliferation to osteoblast differentiation. We directly examined the role of p27 during differentiation of osteoprogenitor cells derived from the bone marrow (BM) of p27-/- mice. BM cells from p27 null mice exhibited increased proliferative activity compared with BM cells from wild-type mice and formed an increased number and larger size of osteoblastic colonies, which further differentiated to the mineralization stage. Although p27-/- adherent marrow cells proliferate faster, they retain competency for differentiation, which may result, in part, from observed higher p21 levels compared with wild type. Histological studies of p27-/- bones also showed an increased cellularity in the marrow cavity compared with the p27+/+. The increased proliferation in bone does not lead to tumorigenesis, in contrast to observed adenomas in the null mice. Taken together, these findings indicate that p27 plays a key role in regulating osteoblast differentiation by controlling proliferation-related events in bone cells.

Animals↗

PubMed1999

Calcitonin gene-related peptide (CGRP) increases intracellular free Ca2+ concentrations but not cyclic AMP formation in CGRP receptor-positive osteosarcoma cells (OHS-4).

Although calcitonin gene-related peptide (CGRP) may act as a local factor in bone, its mechanisms of action on osteoblasts are not well understood. We previously showed the presence of CGRP transcripts and peptide in human OHS-4 osteoblastic cells. The authors investigated the expression of CGRP receptor (CGRP-R) and its intracellular signalling properties in OHS-4 cells. Semi-quantitative RT-PCR analysis showed that OHS-4 cells express much more CGRP-R than calcitonin (CT)-R transcripts. After amplification of CGRP-R by RT-PCR and cloning of amplified fragments, the predicted CGRP-R sequence in OHS-4 cells was found to share 100% identity with human lung CGRP-R. Biochemical analysis showed that hCGRP did not increase intracellular cAMP levels in synchronized OHS-4 cells whatever was the cell cycle position. However, adenylate cyclase activity was functional, as human parathyroid hormone increased cAMP levels. In contrast, hCGRP induced a rapid, transient and dose-dependent increase in free cytosolic calcium levels. The data show that CGRP increases intracellular free Ca2+concentration but is not coupled to adenylate cyclase in CGRP receptor-positive OHS-4 osteosarcoma cells, suggesting that CGRP induces downstream events driven by phospholipase C in these cells.

Animals↗

PubMed1998

Activation of phospholipase C-beta1 via Galphaq/11 during calcium mobilization by calcitonin gene-related peptide.

Interaction of calcitonin gene-related peptide (CGRP) with its receptors leads to stimulation of adenylyl cyclase and/or phospholipase C (PLC). While regulation of adenylyl cyclase is thought to involve the G-protein Gs, it is not known whether activation of PLC results from coupling the receptor to Gq family proteins or whether beta gamma subunits released from receptor-activated Gs activate PLC. We used human bone cells OHS-4 bearing CGRP receptors in which CGRP activates only the PLC signaling pathway to determine how CGRP acts. CGRP increased the concentration of intracellular calcium ([Ca2+]i) within 5 s via a Ca2+ influx through voltage-gated calcium channels and by mobilizing calcium from the endoplasmic reticulum. The activation of effectors, like PLC coupled to G-proteins, is the early event in the pathway leading to inositol 1,4,5-trisphosphate formation, which is responsible for Ca2+ mobilization. Western blotting demonstrated a range of PLC-beta isoforms (beta1, beta3, beta4, but not beta2) and G-proteins (Galphaq/11 and Galphas). Only phospholipase C-beta1 is involved in the mobilization of Ca2+ from the endoplasmic reticulum of Fura-2-loaded confluent OHS-4 cells and the formation of inositol 1,4,5-trisphosphate by CGRP; PLC-gamma have no effect. Activation of PLC-beta1 by CGRP involves the Galphaq/11 subunit, which is insensitive to pertussis toxin, but not Gbeta gamma subunits. We therefore believe that CGRP causes the activation of two separate G-proteins.

Adenylate Cyclase Toxin↗

PubMed1998

[Results of BCG in the treatment of pTa and pT1 bladder tumors. Evaluation of a long protocol using 75 mg of Pasteur strain BCG].

OBJECTIVES: Evaluation of a protocol of intravesical BCG therapy using 75 mg of Pasteur strain BCG with 2 years of maintenance treatment, and a follow-up of up to 60 months. MATERIAL AND METHODS: 189 patients treated by transurethral resection (TUR) for a pTa (N = 80) or pT1 (N = 109) bladder tumour were included in the study. The local and general safety was excellent. We retrospectively compared this series to a group of patients treated by TUR alone (N = 42) another group treated with TUR and Mitomycin C (MMC) (N = 81). The 3 groups were statistically comparable. RESULTS: At 48 months, 62% of patients treated with BCG were recurrence-free, versus only 18% for patients treated with TUR alone and 38% for patients treated with TUR and MMC (p = 0.001). At 42 months, 11% of pT1 tumours treated with BCG had progressed to invasive carcinoma, and this progression occurred during the first 18 months in every case. In comparison, this progression was observed in 25% of pT1 tumours treated by TUR alone and 21% of tumours treated with TUR and MMC. CONCLUSIONS: Our study confirms the efficacy of our BCG protocol ro reduce the potential for recurrence and progression of superficial bladder tumours, despite reduction of the dose to 75 mg. It also suggests the superiority of BCG compared to MMC in terms of recurrence and progression.

Actuarial Analysis↗

PubMed1997

Expression of the CT/CGRP gene and its regulation by dibutyryl cyclic adenosine monophosphate in human osteoblastic cells.

There is general agreement that calcitonin (CT) inhibits bone resorption by its effects on osteoclast function. CT was also found to have direct effects on osteoblast-like cells. In this study, we investigated the expression of CT and calcitonin gene-related peptide (CGRP), the two peptides encoded by the CT/CGRP gene, in human osteosarcoma cell lines and in normal human trabecular osteoblastic cells (HOB), and we studied the modulation of CT/CGRP gene expression by dibutyryl cyclic adenosine monophosphate ((Bu)2, cAMP), a cAMP analog. We first detected by Northern blot hybridization the presence of CT and CGRP mRNAs in different osteosarcoma cell lines (OHS-4, MG-63, Saos-2, HOS-TE85) and HOB cells. In the steady state, OHS-4 cells express slightly more CT and CGRP mRNAs than other cell lines or normal human osteoblasts, in parallel with messengers of differentiated osteoblasts, such as osteocalcin (OC) and alkaline phosphatase (ALP). OHS-4 cells also express CT and CGRP proteins, as demonstrated by immunocytochemistry. Stimulation of OHS-4 cells with 1 mM (Bu)2 cAMP induced a significant increase in mRNA levels for CT (x 2.5) and CGRP (x 3), as determined by a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) procedure. The involvement of a transcriptional mechanism in this effect was evidenced by nuclear run-off transcription assay. In addition, (Bu)2 cAMP increased OC (x 4) and ALP (x 3) mRNA levels in OHS-4 cells. These effects were observed at 24 h and were maximal at 48 h, indicating that (Bu)2, cAMP induced cell differentiation and increased the transcription of the CT/CGRP gene in OHS-4 osteoblast-like cells. The results indicate that human osteosarcoma cells and primary human osteoblastic cells express CT and CGRP mRNA and proteins, and that (Bu)2 cAMP, an activator of protein kinase A, induces up-regulation of osteoblastic phenotypic genes and enhances CT and CGRP gene transcription, indicating that induction of osteoblastic differentiation by (Bu)2 cAMP is associated with enhanced expression of CT and CGRP in human osteoblastic cells.

Alkaline Phosphatase↗