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M Kassem

Publications and source records attributed to M Kassem.

At least 19 recordsLinked to original sources
PubMed2000

Production and action of transforming growth factor-beta in human osteoblast cultures: dependence on cell differentiation and modulation by calcitriol.

BACKGROUND: Transforming growth factor beta (TGF-beta) plays an important role in skeletal remodelling. However, few studies have examined its effects on cultured human osteoblasts. Our aim is to characterise the biological effects of TGF-beta1 on human osteoblasts and to examine the interaction between TGF-beta1 and calcitriol. DESIGN: In vitro study employing two models of normal human osteoblasts: human bone marrow stromal cells [hMS/(OB)] containing osteoprogenitor cells and trabecular bone osteoblasts (hOB), which are mature osteoblasts. A reverse-transcriptase-polymerase-chain-reaction assay was employed to measure steady state mRNA levels of TGF-beta(s) isoforms and receptors. Effects of short-term treatment of TGF-beta1 on osteoblast proliferation and differentiation markers were assessed. The effect of cotreatment of calcitriol (10-8 M) and TGF-beta1 on osteoblast differentiation was also determined. RESULTS: Both hMS(OB) and hOB cells expressed mRNA transcripts of TGF-beta1, TGF-beta2, TGF-beta 3, TGF-beta type I and type II receptors. TGF-beta 1 stimulated osteoblast proliferation in hMS(OB) and in hOB cultures. In hOB cultures, TGF-beta1 stimulated AP production and cotreatment with calcitriol induced a synergistic increase in AP levels to 250 +/- 61% of calcitriol-treated controls. Effects of TGF-beta1 and calcitriol were less pronounced in hMS(OB) cultures. TGF-beta1 inhibited collagen type I production in hMS(OB) cells and these effects were abolished in presence of calcitriol. In presence of calcitriol, TGF-beta1 increased collagen type I production in hOB cells. In both hOB and hMS(OB) cultures, TGF-beta1 inhibited osteocalcin production. CONCLUSIONS: TGF-beta increases osteoblastic cell proliferation irrespective of the differentiation state. In presence of calcitriol, it initiates osteoblast cell differentiation and matrix formation. As TGF-beta inhibits osteocalcin production, other factors are necessary for inducing terminal differentiation of osteoblasts. The observed effects of TGF-beta on human osteoblasts in vitro may represent important regulatory steps in controlling osteoblast cell proliferation and differentiation in vivo.

Activin Receptors, Type I↗
PubMed2000

CBFA1 and topoisomerase I mRNA levels decline during cellular aging of human trabecular osteoblasts.

In order to understand the reasons for age-related impairment of the function of bone forming osteoblasts, we have examined the steady-state mRNA levels of the transcription factor CBFA1 and topoisomerase I during cellular aging of normal human trabecular osteoblasts, by the use of semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). There is a progressive and significant reduction of the CBFA1 steady-state mRNA level down to 50% during cellular aging of human osteoblasts. In comparison to the normal cells, human osteosarcoma cell lines SaOS-2 and KHOS/NP, and the SV40-transformed human lung fibroblast cell line MRC5V2 have 20 to 40% higher levels of CBFA1 mRNA. Similar levels of CBFA1 mRNA are detectable in normal human skin fibroblasts, and these cells also exhibit an age-related decline to the same extent. In addition, the expression of topoisomerase I is reduced by 40% in senescent osteoblasts, and the mRNA levels are significantly higher (40-70%) in transformed osteoblasts and fibroblasts. These changes in gene expression may be among the causes of impaired osteoblast functions, resulting in reduced bone formation during aging.

Adult↗
PubMed2000

Familial isolated hyperparathyroidism as a variant of multiple endocrine neoplasia type 1 in a large Danish pedigree.

We report here our genetic findings of a family in which 14 members were affected with isolated primary hyperparathyroidism. Hyperparathyroidism is the main feature of multiple endocrine neoplasia type 1 (MEN1), making the recently cloned MEN1 gene a prime candidate gene in this family. Significantly positive lod scores were achieved with D11S4946 (3.36) and D11S4940 (3.53), and by combining the results from these two markers, a maximum positive lod score of 4.12 at recombination fraction 0.00 was obtained. Mutation analysis of MEN1 performed by full sequencing identified a missense mutation in exon 4, causing an amino acid change from glutamine to proline at codon 260. This mutation (Q260P) was present in all affected family members, and the inheritance of the mutation was in complete agreement with the disease-associated haplotype. In comparison with the recent functional studies of the menin protein interactions, this mutation is located in a region with little or no binding activity to JunD and activating protein-1 transcription factor. We conclude that some of the familial isolated primary hyperparathyroidism families constitute a milder variant of MEN 1, which is associated with a functionally milder missense mutation.

Adult↗
PubMed1999

Telomere shortening during aging of human osteoblasts in vitro and leukocytes in vivo: lack of excessive telomere loss in osteoporotic patients.

We have compared the telomere length, as assessed by Southern analysis, of telomere restriction fragments (TRFs) generated by RsaI/HinfI digestion of genomic DNA in: (i) in vitro cultured human trabecular osteoblasts undergoing cellular aging; and (ii) peripheral blood leukocytes (PBL) obtained from three groups of women: young (aged 20-26 years, n = 15), elderly (aged 48-85 years, n = 15) and osteoporotic (aged 52-81 years, n = 14). The mean TRF length in human osteoblasts undergoing aging in vitro decreased from an average of 9.32 kilobasepairs (kb) in middle-aged cells to an average of 7.80 kb in old cells. The rate of TRF shortening was about 100 bp per population doubling, which is similar to what has been reported for other cell types, such as human fibroblasts. Furthermore, there was a 30% decline in the total amount of telomeric DNA in senescent osteoblasts as compared with young cells. In the case of PBL, TRF length in the DNA extracted from young women was slightly longer (6.76 +/- 0.64 kb) than that from a group of elderly women (6.42 +/- 0.71 kb). A comparison of TRFs in the DNA extracted from the PBL from osteoporotic patients and from age-matched controls did not show any significant differences (6.47 +/- 0.94 versus 6.42 +/- 0.71 kb, respectively). Therefore, using TRF length as a marker for cellular aging in vitro and in vivo, our data comparing TRFs from osteoporotic patients and age-matched controls do not support the notion of the occurrence of a generalized premature cellular aging in osteoporotic patients.

Adult↗
PubMed1999

Hormone replacement therapy prevents osteoclastic hyperactivity: A histomorphometric study in early postmenopausal women.

In a randomized, double blind, clinical prospective trial comprising 35 women treated with either hormone replacement therapy (HRT) (cyclic estradiol/norethisterone acetate) or placebo we performed histomorphometric studies on paired bone biopsies obtained before and after 2 years of treatment. Untreated women developed a progressively more negative balance at individual bone multicellular units (BMUs) (i.e., wall thickness-erosion depth) (2.2 +/- 1.7 microm vs. -5.7 +/- 1.4 microm; p < 0.01), while women on HRT displayed preservation of bone balance (2.4 +/- 2.4 microm vs. 2.5 +/- 2.5 microm; NS). No significant differences in wall thickness between the two groups were demonstrable, but the untreated women developed a pronounced increase in erosion depth over 2 years (46.9 +/- 1.8 microm vs. 52.0 +/- 1.9 microm; p < 0.05), while the HRT group revealed no change (47.8 +/- 2.7 microm vs. 44.6 +/- 1.7 microm; NS). Furthermore, the placebo group displayed an increased osteoclastic erosion depth (17.8 +/- 1.6 microm vs. 25.0 +/- 1.7 microm; p < 0.001), compared with unchanged values in the HRT group (20.0 +/- 1.6 microm vs. 16.9 +/- 1.4 microm/day; NS). While the placebo group revealed a slight increase in volume referent resorption rate (35 +/- 8% vs. 38 +/- 8%; NS) the HRT group revealed a pronounced decrease (46 +/- 8% vs. 28 +/- 5%; p < 0.05). No significant changes in marrow star volume (an index of trabecular perforations) were demonstrable in either group. Our results demonstrate that bone remodeling in early postmenopausal women is characterized by progressive osteoclastic hyperactivity, which is reduced by cyclic HRT. This reduction of resorptive activity at the BMU level after HRT seems to precede the reduction in activation frequency demonstrated in previous studies on older postmenopausal women.

Bone Remodeling↗
PubMed1998

Cloning and identification of genes that associate with mammalian replicative senescence.

Cellular senescence and limited proliferative capacity of normal diploid cells has a dominant phenotype over immortality of cancerous cells, suggesting its regulation by the expression of a set of genes. In order to isolate the genes that associate with senescence, we have employed a clonal system of conditional SV40 T antigen rat embryo fibroblast cell lines which undergo senescence upon T antigen inactivation. Construction of cDNA libraries from two conditional cell lines and application of differential screening and subtractive hybridization techniques have resulted in the cloning of eight senescence-induced genes (SGP-2/Apo J, alpha 1-procollagen, osteonectin, fibronectin, SM22, cytochrome C oxidase, GTP-alpha, and a novel gene) and a senescence-repressed gene (FRS-2). Three of these genes encode for extracellular matrix proteins, others are involved in the calcium-dependent signal transduction pathways, while the SGP-2/Apo J gene may have a cellular protective function. RNA analysis has shown that the senescence-associated genes are overexpressed in both normal rat embryonic fibroblasts and human osteoblasts cell cultures undergoing aging in vitro. In comparison, the expression of these genes in a rat fibroblast immortalized cell line (208F cells) was down-regulated after both its partial and its full transformation by ras oncogenes. Thus, cloning of senescence-associated genes opens up new ways to elucidate and/or to modulate aging and cancer.

Aging↗
PubMed1998

Estrogen effects on insulin-like growth factor gene expression in a human osteoblastic cell line with high levels of estrogen receptor.

Insulin-like growth factors (IGF)-I and IGF-II are produced by osteoblasts and are important paracrine/autocrine regulators of osteoblast proliferation and differentiation. Estrogen has been reported to increase gene expression of IGF-I in rodent osteoblasts. However, because species differences have been demonstrated in expression of various aspects of the IGF system in bone cells, it is not known whether this action also occurs in human osteoblasts. Thus, we assessed the effects of estrogen treatment on IGF-I and IGF-II gene expression in vitro in a recently developed human fetal osteoblast cell line that has high levels of estrogen receptors. As assessed by a quantitative reverse transcriptase-polymerase chain reaction method, treatment of hFOB/ER9 cells with 17beta-estradiol (E2) increased steady state levels of IGF-I mRNA in a time- and dose- dependent fashion with a maximal increase of 319% +/- 33% (P < 0.01) of control occurring after treatment with 10(-7) M E2 for 48 hours. In contrast, E2 did not alter steady state levels of IGF-II mRNA. The pure (type 2) antiestrogens ICI 182,780 (10(-7) M) and ICI 164,384 (10(-6) M) blocked the E2- induced increase in IGF-I mRNA levels. Interestingly, 4-hydroxytamoxifen (10(-7) M), a documented pure antiestrogen in reproductive tissues, also increased IGF-I mRNA to levels similar to those observed in E2-treated cells. Since E2 was shown to mediate its effects on some target genes through a cAMP-dependent pathway, we studied the interaction between E2 and agents that are known to increase intracellular cAMP. Forskolin (10(-8) M) and dibutyryl cAMP (10(-3) M) increased IGF-I mRNA levels sixfold, and cotreatment with E2 did not affect these changes, consistent with a possible mediation of the estrogen effect on IGF-I gene expression by cAMP. We conclude that in human osteoblastic cells, the IGF-I gene is a target for estrogen action, suggesting that IGF-I may mediate part of the effects of estrogen in human bone.

Bucladesine↗
PubMed1998

Effects of growth hormone treatment on serum levels of insulin-like growth factors (IGFs) and IGF binding proteins 1-4 in postmenopausal women.

BACKGROUND AND OBJECTIVE: Insulin-like growth factor binding proteins (IGFBPs) modulate the actions and bioavailability of insulin-like growth factors (IGFs), however, their regulation in vivo is incompletely understood. In this study we investigated the effects of different doses of growth hormone (GH) on circulating levels of IGFs and IGFBPs. DESIGN: The study was double-blind and placebo-controlled. Patients were treated with either GH in doses of 0.05, 0.10, or 0.20 lU/kg/day of placebo for one week. PATIENTS: Forty post-menopausal women aged 52-73 years with low bone mass. MEASUREMENTS: Serum IGF-I and IGF-II were measured by RIA while IGFBP-1-3 were measured by Western ligand blot (WLB) and compared with determinations by specific immunoassays. IGFBP-4 was measured by WLB alone. RESULTS: Both IGF-I (P < 0.001) and IGF-II (P < 0.01) increased significantly during GH treatment. Additionally, IGFBP-1 (P < 0.001) and IGFBP-2 (P < 0.001) decreased significantly while IGFBP-3 (P < 0.001) and IGFBP-4 (P < 0.05) increased all in a dose-dependent manner. Stepwise (backwards) multiple regression analyses showed that the changes in IGF-I and IGF-II, and age correlated with the change in serum IGFBP-1. Both GH-dosage, the increase in IGF-II, and body mass index correlated with the decrease in IGFBP-2. Furthermore, the increase in serum IGF-I, IGF-II, and triiodothyronine correlated with the increase in IGFBP-3. Moreover, GH-dosage correlated with the increase in serum IGFBP-4. CONCLUSION: GH significantly increased serum IGF-I, IGF-II, IGFBP-3, and IGFBP-4 and decreased serum IGFBP-1 and IGFBP-2 in post-menopausal women.

Aged↗
PubMed1998

The effects of IGF-I and IGF-II on proliferation and differentiation of human osteoblasts and interactions with growth hormone.

BACKGROUND: We have previously shown that growth hormone (GH) consistently stimulates proliferation of human osteoblasts in vitro. In rat osteoblasts, GH augments the effects of insulin-like growth factor (IGF) I on cell proliferation and differentiation. We therefore investigated the effects of IGF-I and -II alone and in combination with GH on human osteoblasts in vitro. METHODS: Human osteoblast-like cells (HOB) were established from trabecular explants (n = 18) and human marrow stromal cells (HMS) from marrow aspiration (n = 21). The cell cultures were stimulated with IGF-I or IGF-II (1, 10 or 100 ng mL-1) alone, in combination with hGH (100 ng mL-1) or after prestimulation with hGH. RESULTS: IGF-I alone, in combination with hGH and after pretreatment with hGH, increased proliferation of HOB and HMS by 49-190% (P < 0.05-0.01). IGF-II alone, in combination with hGH and after pretreatment with hGH increased proliferation of HOB by 57-158% (P < 0.01). In HMS only IGF-II in combination with hGH and after prestimulation with hGH increased proliferation. IGF-I alone and in combination with hGH decreased alkaline phosphatase (AP) in both cell types. IGF-II did not affect AP in HOB, but increased AP in HMS, this effect was abolished by hGH. In HOB, collagen production (PICP) was increased by IGF-II but unaffected by IGF-I. In HMS, PICP was decreased by IGF-I and -II but increased by hGH. Co-stimulation further increased PICP. CONCLUSION: IGF-I and -II exerted proliferative effects on both HOB and HMS. Co-stimulation with GH exhibited synergism in enhancing the proliferative response. In HMS prestimulation improved the proliferative response significantly. The effects of the IGFs on differentiation are more complex and dependent on cell maturation and of the IGF used.

Alkaline Phosphatase↗
PubMed1998

Long-term oral pamidronate treatment inhibits osteoclastic bone resorption and bone turnover without affecting osteoblastic function in multiple myeloma.

This study was performed as a cross-sectional substudy to the Danish-Swedish Pamidronate Study, a randomized placebo-controlled multicentre trial in multiple myeloma. The purpose was to evaluate the biological effects of long-term treatment with oral pamidronate 300 mg daily on bone metabolism by using histomorphometry and analysis of cytokines and biochemical markers of bone turnover. Sixteen patients were included after median 27.5 months of protocolized treatment; 10 patients received active treatment and 6 patients placebo. When compared with placebo, pamidronate treatment was associated with: (a) marked decreased osteoclastic resorption rate (0.86+/-0.59 microm/d vs. 5.7+/-5.0 microm/d, p=0.002), and diminished activation frequency (0.20+/-0.18 yr(-1) vs. 0.72+/-0.55 yr(-1), p=0.014); (b) compensatory reduced volume referent bone formation rate (0.17+/-0.21 yr(-1) vs. 0.71+/-0.54 yr(-1), p=0.007), but unaltered mineral appositional rate; (c) neutral (-0.66+/-5.6 mm) vs. negative (-2.15+/-2.2 microm, p=0.013) bone balance per remodelling cycle; (d) higher trabecular bone volume (21.0+/-6.2% vs. 13.0+/-3.7%, p=0.01); (e) suppressed urinary excretion and serum levels of some of the biochemical markers of bone metabolism; and (f) significant reduction of circulating soluble interleukin-6 receptor (IL-6sR) (25.9+/-4.1 ng/ml vs. 32.1+/-6.6 ng/ml, p=0.04), and (g) a uniform tendency of lower serum and marrow plasma levels of IL-6, IL-1beta, and TNFalpha. Thus oral pamidronate was absorbed in biologically active amounts, and reduced overall bone resorption and bone turnover without impairing osteoblastic bone formation. The observation that cytokine and cytokine receptor levels were reduced extends the possible and potential beneficial actions of bisphosphonates in multiple myeloma.

Administration, Oral↗
PubMed1997

Demonstration of cellular aging and senescence in serially passaged long-term cultures of human trabecular osteoblasts.

The proliferative capacity and cellular and biochemical characteristics of human trabecular bone osteoblasts were analysed throughout their replicative lifespan in vitro. Like several other cell types, human osteoblasts demonstrated a typical Hayflick phenomenon of cellular aging comprising a period of rapid proliferation until cumulative population doubling level (CPDL) 22 to 24, followed by a phase of slow growth and the final cessation of cell division at CPDL 32 to 34. Comparing young cells (less than 20% lifespan completed) and old cells (more than 90% lifespan completed) revealed a progressive increase in population doubling (PD) time, a decrease in attachment frequency, a decrease in the number of S-phase positive cells, a decrease in the rates of DNA, RNA and protein synthesis, an increase in the protein content per cell and an increased proportion of senescence-specific beta-galactosidase positive cells. While osteoblastic production of collagen type I decreased progressively during aging, alkaline phosphatase activity dropped rapidly after the first few passages and then remained constant during the rest of the proliferative lifespan, Significant morphological changes from thin and spindle-shaped early passage young cells to large, flattened and irregularly shaped late passage old cells full of intracellular debris were observed. In comparison, osteoblasts established from an osteoporotic bone sample showed a maximum CPDL of less than 5, had a longer PD time and exhibited abnormal senescent morphology. Thus, we have demonstrated for the first time that human osteoblasts, like several other diploid cell types, have a limited proliferative capacity in vitro and undergo aging and senescence as measured by various cellular and biochemical markers. In addition, preliminary studies show that cells from osteoporotic bone have a severely reduced proliferative capacity. This model of bone cell aging facilitates study of the molecular mechanisms of osteoblast senescence as well as factors related to osteoblast dysfunction in patients with osteoporosis.

Adolescent↗
PubMed1997

Cellular and molecular effects of growth hormone and estrogen on human bone cells.

The aim of the present thesis is to examine some aspects of the biological effects of growth hormone (GH) and estrogen on bone cells in vitro. The first part of the thesis describes characterization of model systems to study normal human osteoblasts and osteoclasts in vitro. Three culture systems for human osteoblasts have been characterized, representing different stages of osteoblasts differentiation/maturation: (1) mature osteoblasts cultured from trabecular bone explants (trabecular osteoblasts), (2) less mature osteoblasts (stromal osteoblasts) cultured from bone marrow and (3) osteoblast precursor cells cultured also from bone marrow. This classification is based on quantitative and qualitative differences in the expression of osteoblast phenotypic markers by these cells. These systems are useful in studying the regulation of hormones and growth factors of different stages of osteoblast differentiation. Further characterization of the osteoblast differentiation pathway is still needed, especially the identification of surface markers that can definitively identify intermediate stages of osteoblast differentiation. Normal human osteoclasts were cultured from bone marrow mononuclear cells and exhibited the main characteristics of osteoclast phenotype: production of tartrate-resistant acid phosphatase, presence of a ruffled border and the ability to resorb mineralized matrix. This model is useful for studying factors regulating osteoclast commitment and differentiation. The second part of the thesis deals with the effects of GH on proliferation and differentiation of trabecular and stromal osteoblasts. Effects of GH on human osteoblasts were dependent on their degree of maturation. GH stimulated cell proliferation in both trabecular and stromal osteoblasts. While it increased the functional activity of trabecular osteoblasts, these effects were absent in stromal osteoblast cultures. Human trabecular osteoblasts produce mainly IGF-II, IGFBP-3 and minute quantities of IGF-I in culture. GH does not seem to regulate the local production of IGF-II or IGFBP-3. However, IGFs and their binding proteins may exert important regulatory effects on the biological effects of GH on human osteoblasts, and this role needs to be studied. Sincer GH exerted profound effects on the biological functions of human osteoblasts in vitro, the hypothesis that either decreased production of GH or decreased sensitivity of bone cells to its action leads to bone loss and osteoporosis was examined. No differences in the basal or GH-stimulated production of IGF-I, IGF-II or IGFBP-3 were found between osteoporotic patients and age-matched normals. Similarly, The response of bone cells to in vivo and in vitro stimulation by GH was similar in the two groups. These studies do not support the hypothesis of the presence of major defects in production of GH or the presence of resistance to its effects in bone cells in patients with osteoporosis. The last part of the thesis deals with the cellular mechanisms mediating estrogen actions on bone cells. Thus, the potential role of estrogen regulation of bone-resorbing cytokines in the pathogenesis of bone loss in postmenopausal women was examined. Estrogen was found to inhibit IL-6 production and gene expression in an immortalized human osteoblastic cell line expressing high levels of estrogen receptors (hFOB/ER9 cell line). To further examine the relationship between estrogen deficiency and early postmenopausal bone loss, levels of IL-6, IL-6sR, IL-1 alpha, IL-1 beta and IL-1ra were measured in bone marrow plasma and bone marrow cultures obtained from 40 postmenopausal women, half of whom were on estrogen replacement therapy. No difference was found between the groups in any of these parameters. This suggests that neither IL-1 nor IL-6 by itself is the major mediator for increased bone loss due to estrogen deficiency in the early postmenopausal period. (ABSTRACT TRUNCATED)

Bone and Bones↗
PubMed1997

[Apoptosis: molecular aspects].

Many signals and external stimuli regulate the apoptosis activity by interaction with the genome. These stimuli include morphogenetic signals, physiological factors, and environmental influence. The signals mediate their effect on cells with suitable receptors, relevant signalling pathways, and competence to execute the apoptosis cascade. Apoptosis is triggered indirectly by deprivation of survival factors, or directly by intercellular cell death signalling factors, and also by unbalanced intracellular messenger molecules, which are, more or less, involved in regulation of both programmed cell death and survival. Several genes are involved in regulation of cell survival and apoptosis: bcl-2/bax, p53, c-myc and transcription factors such as cdk, c-myc, c-fos and c-jun. Apparently, apoptosis could be triggered by increased or inhibited gene expression as well as biochemical reactions without changed gene expression. The morphological changes during apoptosis reflect a cascade of genetic and biochemical reactions in the cell. In the signal transduction pathway both secondary messenger Ca2+, different kinases, and polyamines are involved. Cysteine proteases cleave cytoskeletal proteins, endonucleases divide DNA into fragments, and transglutaminases cross-link macromolecules. Degradative enzymes such as proteases, endonucleases and transglutaminases are activated during apoptosis, leading to cellular collapse and formation of vesicular apoptotic bodies. Both increased and inhibited apoptosis activity may have pathological consequences. New therapeutic strategies aim to counteract dysregulation of apoptosis in specific tissues by pharmacological intervention. Thus there is a need for identification of molecules and gene products involved in regulation of apoptosis activity and clarification of the conditions where this knowledge may be used.

Apoptosis↗
PubMed1996

Estrogen inhibits interleukin-6 production and gene expression in a human osteoblastic cell line with high levels of estrogen receptors.

Some studies suggest that estrogen acts on bone by decreasing the production of interleukin-6 (IL-6), a cytokine that increases bone resorption, by osteoblasts or bone marrow cells. However, other studies have not confirmed this, possibly because of a low and variable number of estrogen receptors (ER) in the model systems used. Thus, we employed a recently developed human fetal osteoblast cell line with high levels of ER. Treatment (n = 4 experiments) with 0.01 to 10 nM of 17 beta-estradiol had no effect on the constitutive production of IL-6. However, stimulated production, induced by treatment with IL-1 beta plus tumor necrosis factor-alpha (TNF-alpha), was reduced in a dose-dependent manner to 74 +/- 3% (mean +/- SEM) of control (p < 0.01). This response was blocked by cotreatment with the type II antiestrogen ICI 182,780. Treatment with hydrocortisone (1 microM), a known inhibitor of IL-6 production in many cell types, reduced IL-6 production to 17 +/- 1% of control (p < 0.001). As assessed by Northern analysis, treatment (n = 3 experiments) with 0.01-10 nM of 17 beta-estradiol decreased steady-state levels of IL-6 mRNA in a dose-dependent manner. These data support the hypothesis that at least part of the antiresorptive action of estrogen in humans is mediated by decreased production of IL-6 by osteoblastic cells.

Base Sequence↗
PubMed1996

Isolation and characterization of osteoblast precursor cells from human bone marrow.

Osteoblasts are derived from precursor cells present in low frequency in the stromal element of bone marrow. Because of the lack of a practical procedure to isolate osteoblast precursors from early cultures of plastic adherent cells from bone marrow, previous studies of marrow stromal cells have been made in confluent cultures of bone marrow when the osteoblast (OB) precursors are already differentiated. Also these studies utilized cultures containing mixed populations of cells including hematopoietic cells. Thus we have employed a negative immunoselection procedure to remove contaminating hematopoietic cells and to isolate nearly homogeneous populations of early human stromal cells derived from the plastic-adherent mononuclear marrow cells cultured in the presence of serum. By reverse transcriptase polymerase chain reaction (RT-PCR) analysis for mRNA, and by immunocytochemical study for protein, we studied the sequential expression in culture of multiple markers of the osteoblast phenotype--alkaline phosphatase, osteopontin, parathyroid hormone receptor, types I and III procollagen, and osteocalcin--as well as lipoprotein lipase (LPL), a marker of the adipocyte phenotype. At an early stage of culture (7-9 days), human OB precursors formed colonies of variable sizes that expressed low levels of mRNA and protein concentrations of OB markers, and their concentration increased on growth to a confluent monolayer (approximately 14 days). LPL mRNA was expressed at high levels in the colony stage, and its level decreased upon confluency, suggesting a loss of potential for commitment to the adipocyte lineage. Interestingly, treatment with dexamethasone at 10(-8) M increased the expression for some of the osteoblast markers and for the LPL gene and was required for the deposition of mineralized matrix and for the formation of adipocytes containing cytoplasmic lipid droplets in confluent cultures. Cloned single early colonies were able to coexpress the osteoblast and adipocyte markers (as assessed by RT-PCR). Thus these immunoselected marrow stromal cells have the characteristics of authentic human osteoblast precursor cells which also are capable of differentiating into adipocytes.

Alkaline Phosphatase↗
PubMed1996

European and North American Experience with HRT for the prevention of osteoporosis.

Hormone replacement therapy (HRT) has been the method of choice for the prevention of postmenopausal osteoporosis since the early 1990s. Although a number of routes of administration are now available, HRT is still predominantly administered orally. In the United States, HRT formulations traditionally comprise conjugated equine estrogens. In Europe, however, HRT preparations tend to be based on 17 beta-estradiol, a natural human estrogen. Furthermore, distinct patterns of HRT use are apparent based on the age of the woman receiving it. Current recommendations are that early postmenopausal women (in their early 50s) receive sequential combined estrogen/progestogen therapy with continued monthly bleeds, while in women who are at least 1 year postmenopausal, continuous combined HRT, which leads to endometrial atrophy and cessation of monthly bleeding, is preferred. Clinical experience to date clearly demonstrates that long-term HRT unequivocally increases bone mass and reduces the risk of fractures in postmenopausal women, with no significant differences between sequential and continuous combined prescribing regimens. Data demonstrating that antiestrogens such as tamoxifen may preserve bone mass have led to the initiation of large-scale trials to determine the potential clinical utility of such agents for the prevention of osteoporosis in postmenopausal women. Nonhormonal therapeutic approaches are now also available, most notably bisphosphonates and vitamin D analogs. At present, however, traditional HRT remains the regimen of choice for the prevention of postmenopausal osteoporosis, given its additional beneficial effects on acute menopausal symptoms, as well as on the cardiovascular system and brain.

Administration, Oral↗