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Biomedical subjects

S Migliaccio

Publications and source records attributed to S Migliaccio.

At least 19 recordsLinked to original sources
PubMed2002

The role of the alphaVbeta3 integrin in the development of osteolytic bone metastases: a pharmacological target for alternative therapy?

Common cancers frequently develop bone metastases, which are often osteolytic in nature due to activation of osteoclast differentiation and bone resorption. This may result from direct stimulation of these cells by the metastasis, or may be due to indirect enhancement of osteoclast activity by osteoblasts. A further feature of the bone metastasis is an extensive medullary angiogenesis which supports tumor growth. The alphaVbeta3 integrin is highly expressed in bone metastatic cells, as well as in osteoclasts and in the activated endothelium, where it plays a major role in cell function. In contrast, this receptor is barely expressed in other cell types. Our hypothesis is that inhibition of this mechanism, which is not widespread in most tissues and at the same time is common to several steps of cancer-induced osteolysis (i.e., homing, growth, and survival of metastatic cells, osteoclast bone resorption, and angiogenesis), should represent a suitable target to block the development of bone spreading of metastatic tumors. We extend this hypothesis to downstream signalling molecules activated by ligation of the alphaVbeta3 integrin, some of which (i.e., Src, PYK2, and Shc) could have similar specific roles in tumor cells, activated endothelium and osteoclasts, but not in other cell types.

Antineoplastic Agents↗
PubMed2002

The selective estrogen receptor modulator raloxifene regulates osteoclast and osteoblast activity in vitro.

Raloxifene is a selective estrogen receptor modulator (SERM) that prevents bone loss. Although it is largely used for the treatment of osteoporosis, the mechanisms by which this compound modulates the activity of bone cells are still poorly understood. In this study we investigate whether raloxifene affects osteoclast and osteoblast activity in vitro. Bone marrow cultures were established from neonatal mice and treated with 1,25(OH)(2) vitamin D(3) (VitD(3), 10(-8) mol/L) to induce osteoclast generation. Similar to 17beta-estradiol, raloxifene significantly reduced the number of osteoclasts in a concentration-dependent manner, with maximal inhibition at 10(-11) mol/L (-48%). However, as for 17beta-estradiol, at a high concentration (10(-7) mol/L), the inhibitory effect of raloxifene was abolished. In a pit assay, raloxifene inhibited bone resorption. A maximal effect was observed at 10(-9) mol/L, and maintained at a high concentration, indicating that inhibition of osteoclast formation and inhibition of bone resorption may be due to activation of, at least in part, different pathways. Osteoblasts from neonatal mice calvariae were also exposed to raloxifene. In these cells, this compound induced a concentration-dependent increase of proliferation, which was blocked by the estrogen-receptor antagonist ICI 164,384. Raloxifene also increased the osteoblast-specific transcription factor Cbfa1/Runx2 and alpha2 procollagen type I chain mRNAs, with a pattern that only partially coincided with that of 17beta-estradiol. Consistent with decreased osteoclastogenesis, raloxifene inhibited the mRNA expression of interleukin (IL)-1beta and IL-6 at a low concentration, but not at a high concentration, whereas 17beta-estradiol had similar effects on IL-6 and inhibited IL-1beta at both concentrations. Furthermore, both compounds were able to inhibit tumor necrosis factor (TNF)-alpha-induced IL-1beta, but not IL-6, increase. In conclusion, these data show that raloxifene negatively modulates osteoclasts, and positively affects osteoblasts, suggesting not only an antiresorptive role, but also an osteoblast stimulatory role.

Animals↗
PubMed2001

Melanoma cells stimulate osteoclastogenesis, c-Src expression and osteoblast cytokines.

Malignant melanomas metastasise to the bone and enhance osteoclast bone resorption. We demonstrated that a 48-h-B16 melanoma cell conditioned media (B16CM) induced osteoclastogenesis in mouse bone marrow cultures, without the requirement of B16 cell-bone marrow cell co-culture. B16 cells transcriptionally expressed detectable levels of TGFbeta1, IL-6, M-CSF, GM-CSF and TNFalpha mRNAs, albeit to a lower extent compared with levels in osteoblasts, and failed to express PTHrP, OPGL, OPG and IL-1beta. Interestingly, B16CM greatly upregulated IL-1beta, IL-6 and GM-CSF, and modestly enhanced TNFalpha and OPGL mRNA expression in osteoblasts, suggesting a potential indirect stimulation of osteoclastogenesis via the osteogenic lineage. B16CM barely upregulated c-Fos, but strongly and time-dependently enhanced c-Src expression in the total bone marrow cultures during osteoclast differentiation. Moreover, c-Src expression was enhanced in differentiated and purified osteoclast preparations to higher levels than in stromal cells. In conclusion, melanoma induces osteoclast generation with a paracrine mechanism independent of cell-cell contact, specifically upregulating c-Src in osteoclasts and cytokine expression in osteoblasts.

Animals↗
PubMed2001

Reduced growth and skeletal changes in zinc-deficient growing rats are due to impaired growth plate activity and inanition.

We investigated the effects of dietary zinc deficiency on skeletal metabolism in an animal model. Thirty 21-d-old male Sprague-Dawley rats were fed for 28 d either a zinc-deficient (ZD) diet (1 mg zinc/kg) or a normal diet ad libitum (AL, 50 mg zinc/kg) or in the same quantity as the ZD (pair-fed, PF). Only in the ZD group were general physical signs of zinc deficiency observed. Compared with the AL and PF rats, ZD rats showed significantly lower mean values in ponderal growth rate, femur weight and length, circulating levels of insulin-like growth factor-I, bone mechanical properties and concentration of zinc and, on histomorphometry, a decrease in the thicknesses of the overall growth plate and hypertrophic cartilage. In contrast, although bone volume was significantly lower in the ZD and PF rats than in the AL rats, no difference was observed between the ZD and PF rats. Osteoclast surface/bone surface and osteoclast number/bone surface ratios were significantly greater in PF rats than in the other two groups and not different in ZD and AL rats. Collectively, these data indicate that zinc deficiency has profound effects on the skeletal system of growing rats. In particular, the effects of zinc deficiency on bone growth and mass are the result of the reduced activity of the growth plate, likely mediated by impairment in the insulin-like growth factor-I system. We did not demonstrate an effect on bone mass via increased bone resorption.

Animals↗
PubMed2001

Apparent cure of a newborn with malignant osteopetrosis using prednisone therapy.

A newborn girl with hemorrhagic purpura, suspected neonatal sepsis, and pale and dry skin was lethargic with remarkable hepatosplenomegaly, convergent strabismus, severe anemia, and elevated alkaline phosphatase activity. Radiographs showed a generalized increase in bone density, small medullary cavities, sclerosis of the skull and vertebrae, transverse wavy stripes of sclerotic bone in the metaphyses, and bone-in-bone appearance in phalanges of hands and feet. On this basis, she was diagnosed with malignant infantile osteopetrosis. On the first day of life, the infant was given a blood transfusion and vitamin K (1 mg intravenously [iv]). Corticosteroid therapy was started with prednisone (2 mg/kg per day). She showed marked improvement of symptoms. On the 26th day and 42nd day of life, she received additional blood transfusions. On the 49th day, the patient was discharged and corticosteroid therapy was continued at a regimen of 5 mg/day. Subsequent blood sample analyses revealed normal values for age. At 1 year of life, a bone marrow sample showed normal white and red cell lineages. X-ray confirmed attenuation of the bone sclerosis; therefore, bone marrow transplantation (BMT) was not implemented. At the age of 1.5 years, prednisone therapy was discontinued gradually and withdrawn before the age of 2 years. Subsequent follow-up showed normalization of all radiological and hematologic parameters. At present, the patient is 3 years old and appears healthy with apparently complete regression of the disease.

Ankle↗
PubMed2000

Decreased c-Src expression enhances osteoblast differentiation and bone formation.

c-src deletion in mice leads to osteopetrosis as a result of reduced bone resorption due to an alteration of the osteoclast. We report that deletion/reduction of Src expression enhances osteoblast differentiation and bone formation, contributing to the increase in bone mass. Bone histomorphometry showed that bone formation was increased in Src null compared with wild-type mice. In vitro, alkaline phosphatase (ALP) activity and nodule mineralization were increased in primary calvarial cells and in SV40-immortalized osteoblasts from Src(-/-) relative to Src(+/+) mice. Src-antisense oligodeoxynucleotides (AS-src) reduced Src levels by approximately 60% and caused a similar increase in ALP activity and nodule mineralization in primary osteoblasts in vitro. Reduction in cell proliferation was observed in primary and immortalized Src(-/-) osteoblasts and in normal osteoblasts incubated with the AS-src. Semiquantitative reverse transcriptase-PCR revealed upregulation of ALP, Osf2/Cbfa1 transcription factor, PTH/PTHrP receptor, osteocalcin, and pro-alpha 2(I) collagen in Src-deficient osteoblasts. The expression of the bone matrix protein osteopontin remained unchanged. Based on these results, we conclude that the reduction of Src expression not only inhibits bone resorption, but also stimulates osteoblast differentiation and bone formation, suggesting that the osteogenic cells may contribute to the development of the osteopetrotic phenotype in Src-deficient mice.

Alkaline Phosphatase↗
PubMed2000

Qualitative assessment of natural apatite in vitro and in vivo.

Among the natural and synthetic materials investigated as bone graft substitutes, much interest has been focused on natural apatite obtained from low temperature heat-deproteinated compact bone. Previous research demonstrates that, when treated at a temperature below 500 degrees C, this material maintains its characteristic ultrastructural features, with a high surface/volume ratio, while as an implant material, it offers the host tissue a large surface of interaction. In vitro and in vivo tests showed that natural apatite is well tolerated and is a good osteoconducing material. The present in vivo study in rabbits was carried out to first investigate the behavior and capacity of natural apatite implants to stimulate bone ingrowth, and then to analyze the cells located at the bone/material interface. Synthetic hydroxyapatite was used as a control material. In a parallel in vitro study, we investigated the activity of differentiated osteoblasts and periosteal cells obtained from rats and new-born rabbits, incubated with natural apatite and synthetic hydroxyapatite. The in vivo study showed that natural apatite allows osteoblasts to form new bone tissue, adhering to the implant with ingrowth into the implant structure. In the presence of synthetic hydroxyapatite, a less pronounced osteoblastic activity was observed. In agreement with these observations, the in vitro study showed that natural apatite is more effective in attracting cells, favoring their proliferation and stimulating alkaline phosphatase activity. These findings suggest that natural apatite is more suitable for bone filling or bone regeneration than synthetic hydroxyapatite.

Animals↗
PubMed2000

Transient estrogen exposure of female mice during early development permanently affects osteoclastogenesis in adulthood.

Estrogens modulate bone tissue turnover in both experimental animal models and postmenopausal women. Our previous studies have shown that exposure to diethylstilbestrol (DES) during the perinatal period increases peak bone mass in female mice in adulthood. We investigated whether developmental DES exposure can influence bone mass by affecting osteoclastogenesis. Female mice were injected with 100 microg/kg body weight DES from days 9-16 of gestation or, alternatively, pups received neonatal injections of 2 microg of DES from days 1-5 of life. Animals were weaned at 21 days of age and effects of estrogen on bone cells were evaluated in adulthood. A significant increase in bone mass in female mice was already observed at 2 months, with a maximal effect in older animals. Bone sections from DES-treated animals showed a significant decrease in osteoclast number and tartrate-resistant acid phosphatase (TRAP) enzymatic activity as compared with controls. To verify the importance of the estrogen surge at puberty in this event, a group of control and DES-treated mice were ovariectomized at 17 days to prevent puberty, and potential effect on osteoclastic cells was evaluated in adulthood. As expected, ovariectomy induced an increase of TRAP-positive cells. DES treatment blunted the ovariectomized-dependent increase of the total number of osteoclastic cells, suggesting a role of developmental DES exposure in the process of bone-cell imprinting. Our data indicate, for the first time, that transient changes in estrogen levels during development modulate bone turnover and osteoclastogenesis likely participating in bone-cell imprinting during early phases of bone development, and that this effect could be induced by direct alteration of bone microenvironment.

Animals↗
PubMed1999

Decreased proliferation and altered differentiation in osteoblasts from genetically and clinically distinct craniosynostotic disorders.

Craniosynostoses are a heterogeneous group of disorders characterized by premature fusion of cranial sutures. Mutations in fibroblast growth factor receptors (FGFRs) have been associated with a number of such conditions. Nevertheless, the cellular mechanism(s) involved remain unknown. We analyzed cell proliferation and differentiation in osteoblasts obtained from patients with three genetically and clinically distinct craniosynostoses: Pfeiffer syndrome carrying the FGFR2 C342R substitution, Apert syndrome with FGFR2 P253R change, and a nonsyndromic craniosynostosis without FGFR canonic mutations, as compared with control osteoblasts. Osteoblasts from craniosynostotic patients exhibited a lower proliferation rate than control osteoblasts. P253R and nonsyndromic craniosynostosis osteoblasts showed a marked differentiated phenotype, characterized by high alkaline phosphatase activity, increased mineralization and expression of noncollagenous matrix proteins, associated with high expression and activation of protein kinase Calpha and protein kinase Cepsilon isoenzymes. By contrast, the low proliferation rate of C342R osteoblasts was not associated with a differentiated phenotype. Although they showed higher alkaline phosphatase activity than control, C342R osteoblasts failed to mineralize and expressed low levels of osteopontin and osteonectin and high protein kinase Czeta levels. Stimulation of proliferation and inhibition of differentiation were observed in all cultures on FGF2 treatment. Our results suggest that an anticipated proliferative/differentiative switch, associated with alterations of the FGFR transduction pathways, could be the causative common feature in craniosynostosis and that mutations in distinct FGFR2 domains are associated with an in vitro heterogeneous differentiative phenotype.

Acrocephalosyndactylia↗
PubMed1999

Mechanisms of osteoclast dysfunction in human osteopetrosis: abnormal osteoclastogenesis and lack of osteoclast-specific adhesion structures.

Osteoclasts from a patient affected by osteopetrosis were examined in vivo and in vitro. Iliac crest biopsy revealed an osteosclerotic pattern, with prominent numbers of osteoclasts noted for hypernuclearity and incomplete adherence to the bone surface. A population comprising tartrate-resistant acid phosphatase (TRAP)-positive, multinucleated and mononuclear cells, and alkaline phosphatase-positive stromal fibroblasts was obtained in vitro from bone marrow. Mononuclear TRAP-positive precursors spontaneously fused in culture to form giant osteoclast-like cells. These cells expressed the osteoclast marker MMP-9 and calcitonin receptor, and lacked the macrophage marker, Fc receptor. Expression and distribution of c-src, c-fms, and CD68, and response to steroid hormones relevant to osteoclast differentiation and function were apparently normal, whereas cell retraction in response to calcitonin was impaired. TRAP-positive multinucleated cells did not form osteoclast-specific adhesion structures (clear zone, podosomes, or actin rings). Bone resorption rate was severely reduced in vitro. Focal adhesions and stress fibers were observed en lieu of podosomes and actin rings. Adhesion structures contained low levels of immunoreactive vitronectin receptor, most of this integrin being retained in cytoplasmic vesicles. These data provide the first characterization of abnormal differentiation and function of human osteopetrotic osteoclast-like cells.

Acid Phosphatase↗
PubMed1998

Modulation of estrogen receptor levels in mouse uterus by protein kinase C isoenzymes.

We have recently shown that protein kinase C (PKC) modifies estrogen receptor (ER) binding and modulates the responsiveness to estrogens in a clonal osteoblast-like cell line stably transfected with the ER. The purpose of the present study was to determine whether the interaction observed between the ER and PKC signaling in these cells occurs in additional estrogen target organs, such as the uterus. When uteri were incubated for 2 h with increasing concentrations of a kinase inhibitor (H7), ER binding was enhanced in a dose-dependent manner. Stimulation of PKC with phorbol ester reduced PKC activity levels, but increased ER binding. Interestingly, the changes in binding appeared to be due primarily to alterations in cytosolic ER levels, as binding in the nuclear fraction was minimally enhanced. When levels of ER messenger RNA were evaluated by Northern blot analysis, no differences were observed among the H7- or 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated and untreated groups. Western blot analysis, however, demonstrated that levels of ER cytosolic protein in the H7-, TPA-, and staurosporine-treated groups were increased relative to those in the untreated controls. When uteri were incubated with diethylstilbestrol in the presence of either H7 or TPA, no change in cytosolic ER levels was found, suggesting that only unoccupied ERs are responsive to modulation by PKC. Western blotting of the various PKC isoforms indicated that although PKC alpha, -beta1, -betaII, -delta, and -zeta are expressed in the uterus, only PKC alpha and -beta1 are translocated from the soluble to the particulate fraction and then degraded after phorbol ester stimulation. Hence, one or both of these latter PKC isoforms may regulate cytosolic ER levels. Collectively, these data indicate that PKC may play an important role in the modulation of uterine ER levels and that PKC may exert its effect on the ER at some posttranscriptional or posttranslational step. Finally, our results show that an ER-PKC interaction occurs in a whole organ such as the uterus and that this interaction may be important in the regulation of the ER activity in a variety of estrogen-responsive tissues.

Animals↗
PubMed1998

Phospholipase D- and protein kinase C isoenzyme-dependent signal transduction pathways activated by the calcitonin receptor.

The calcitonin receptor expressed by the porcine LLC-PK1 renal tubule cells is a seven-transmembrane domain, G protein-coupled receptor activating adenylyl-cyclase and phospholipase C. Salmon calcitonin stimulated dose- and time-dependent release of the phospholipase D-dependent phosphatidylcholine product [3H] choline with an EC50 = 2.5 +/-0.3 x 10(-8) M, similar to that determined for phosphoinositide metabolism (EC50 = 4.5 +/-1.0 x 10(-8)M). The hormone failed to induce release of [3H]phosphocholine and [3H]glycerophosphocholine, ruling out activation of phosphatydilcholine-specific phospholipase C and phospholipase A. Calcitonin stimulated phosphatidic acid, a product of phospholipase D-dependent phosphatydilcholine hydrolysis. Activation of phospholipase D was confirmed by release of [3H]phosphatydilethanol, a specific and stable product in the presence of a primary alcohol. Activation of calcitonin receptor induced diacylglycerol formation, with a rapid peak followed by a prolonged increase, due to activation of phospholipase C and of phospholipase D. Consequently, the protein kinase-C alpha, but not the delta isoenzyme, was cytosol-to-membrane translocated by approximately 50% after 20 min exposure to calcitonin, whereas protein kinase-C zeta, which was approximately 40% membrane-linked in unstimulated cells, translocated by approximately 19%. The human calcitonin receptor expressed by BIN-67 ovary tumor cells, although displaying higher affinity for calcitonin, failed to activate phospholipase D and protein kinase-C in response to the hormone. This receptor lacks the G protein binding consensus site due to the presence of a 48-bp cassette encoding for a 16-amino acid insert in the predicted first intracellular loop. This modification is likely to prevent the calcitonin receptor from associating to phospholipase-coupled signaling.

Animals↗
PubMed1998

Colony stimulating factor-1-induced osteoclast spreading depends on substrate and requires the vitronectin receptor and the c-src proto-oncogene.

The colony stimulating factor 1 (CSF-1) regulates osteoclastogenesis and bone resorption. Mutations in the CSF-1 gene cause an osteopetrosis characterized by the absence of osteoclasts. Mature osteoclasts respond to CSF-1 with inhibition of bone resorption and an increment of cell spreading. Herein we demonstrate that CSF-1-induced osteoclast spreading depends on the substrate the osteoclast interacts with and requires integrity of the vitronectin receptor and of the c-src proto-oncogene. Rabbit osteoclasts were allowed to attach to glass, serum, osteopontin, and bone substrates, and were treated with 10 ng/ml human recombinant CSF-1 for 4 h. In osteoclasts plated on glass, the cytokine induced 70% inhibition of bone resorption and 1.8-fold stimulation of cell spreading, without changes in podosome expression and microfilament array. In contrast, CSF-1 induced a 2.5-fold increase of osteoclasts showing filopodia, and a 9.5-fold increase of osteoclasts presenting lamellipodia, indicating that membrane motility was required for cell spreading. Osteoclasts plated on serum substrates showed a 50% reduction of spontaneous spreading. However, in this circumstance, CSF-1 still stimulated an increase of osteoclast area. In osteoclasts cultured on osteopontin substrate or on bone slices, an inhibition of CSF-1-induced osteoclast spreading was observed. To establish involvement of the vitronectin receptor and c-src proto-oncogene, cells were treated with the alpha vbeta3 integrin neutralizing antibody, LM609, or c-src antisense oligonucleotides, which reduced CSF-1-induced osteoclast spreading by 57% and 60%, respectively. The results demonstrate that CSF-1-induced osteoclast spreading requires both the vitronectin receptor and the c-src proto-oncogene and that this action is modulated by the adhesion substrata.

Actin Cytoskeleton↗
PubMed1997

Transforming growth factor-beta enhances adhesion of melanoma cells to the endothelium in vitro.

Melanoma invasion requires migration through the vascular barrier. An early event in this process is the adhesion of metastatic cells to the endothelium. To elucidate the role of TGF-beta in the regulation of this process, human melanoma SK-MEL24 cells were labelled with [5'-(3)H]-thymidine and co-cultured with bovine pulmonary artery endothelial-cell monolayers. Radioactivity was assumed to be proportional to the number of SK-MEL24 cells bound to the endothelium. A low number of melanoma cells adhered to endothelial cells in a time-related manner. Pretreatment for 24 hr with 0.001 to 10 ng/ml TGF-beta1 or TGF-beta2 of both cell types enhanced melanoma-endothelium adhesion in a dose-dependent manner. Both melanoma and endothelial cells expressed RI- and RII-type TGF-beta receptors. The effect of TGF-beta was abolished by co-incubation with the proteoglycan decorin. Conditioned media from melanoma-endothelium co-cultures contained latent TGF-beta and failed to affect cell-cell adhesion. However, activation of TGF-beta by heating the medium or reducing the pH, increased melanoma-endothelium adhesion to an extent similar to that of the TGF-beta administered to the cultures. Zimography demonstrated that both cell types expressed urokinase-type plasminogen activator (uPA). Addition of plasminogen to the co-cultures, which was likely to be activated to plasmin by uPA, resulted in activation of TGF-beta and parallel stimulation of melanoma-endothelium adhesion. In conclusion, TGF-beta may enhance adhesion of melanoma cells to the endothelium, playing a relevant autocrine/paracrine role in the progression of invasive melanoma.

Activin Receptors, Type I↗
PubMed1996

Alterations of maternal estrogen levels during gestation affect the skeleton of female offspring.

Estrogens have important effects on bone turnover in both humans and experimental animals models. Moreover, the decreased level of estrogen after menopause appears to be one of the key factors in determining postmenopausal osteoporosis. The presence of estrogen receptor in both osteoblasts and osteoclasts has suggested a direct role of these steroid hormones on bone tissue. Thus, this tissue is now regarded as a specific estrogen target tissue. Exposure to estrogens during various stages of development has been shown to irreversibly influence responsive target organs. We have recently shown that transient developmental neonatal exposure (days 1-5 of life) of female mice to estrogen resulted in an augmented bone density in the adult animals. The aim of the present study was to evaluate whether short-term modification of maternal estrogen levels during pregnancy would induce changes in the skeleton of the developing fetuses and to identify any long-term alterations that may occur. Pregnant mice were injected with varying doses (0.1-100 micrograms/kg maternal BW) of the synthetic estrogen diethylstilbestrol (DES) from day 9-16 of pregnancy. Offspring were weaned at 21 days of age, and effects on bone tissue of the female mice were evaluated in adulthood (6-9 months of age). Prenatal DES treatment(s) did not significantly affect BW. However, a dose-dependent increase in bone mass, both in the trabecular and cortical compartments, was observed in the prenatal DES-exposed female offspring. Furthermore, long bones of DES-exposed females were shorter than controls. Normal skeletal mineralization accompanied these changes in the bone tissue, as shown by a parallel increase in skeletal calcium content. Double tetracycline labeling performed in 6-month-old DES-exposed animals showed an increase in mineral apposition rate in adult DES-exposed mice as compared with untreated control animals, although no significant difference in the circulating estrogen levels was found in animals of this age. Experiments were then performed to evaluate whether perturbation of the estrogen surge at puberty in these diethylstilbestrol (DES)-exposed mice could reverse the observed changes. Femur length was chosen as a marker of potential estrogenic effect. Prepubertal ovariectomy of the prenatally DES-treated animals could only partially reverse the effects observed in the skeleton of the DES-treated animals. Further experiments were performed to evaluate whether these changes could have occurred in utero. CD-1 pregnant female mice were injected with DES (100 micrograms/kg maternal BW) from days 9-15 of gestation. On day 16 of gestation, fetuses were examined and stained by a standard Alizarin Red S and Alcian Blue procedure to visualize calcified and uncalcified skeletal tissue. Estrogen treatment induced an increase in the amount of calcified skeleton as compared with untreated controls and also a decrease in the length of long bones, strongly suggesting a change in both endochondral ossification and endosteal and periosteal bone formation. In summary, these data show, for the first time, that alterations in the maternal estrogenic levels during pregnancy can influence early phases of fetal bone tissue development and subsequently result in permanent changes in the skeleton. Finally, the effect of this short-term estrogen treatment can be seen in the fetal skeleton, suggesting an estrogen-imprinting effect on bone cell-programming in fetal life because treatment effects on bone cell turnover can be observed later in adult life.

Animals↗
PubMed1996

Estrogen receptor gene disruption: molecular characterization and experimental and clinical phenotypes.

The estrogen receptor (ER) is thought to play a crucial role in the regulation of many life processes, including development, reproduction and normal physiology. Because there have been no known mutations of the estrogen receptor in normal tissue of humans and animals, its presence and tissue distribution is thought to be essential for survival. Using the techniques of homologous recombination, we have disrupted the ER gene and have produced a line of transgenic mice possessing the altered ER gene (ERKO). The mouse ER gene was disrupted by inserting a 1.8 kb PGK-Neomycin sequence into exon 2, approximately 280 bp downstream of the transcription start codon. The correct targeting of the disruption was demonstrated by Southern blot analysis and PCR. Western blot analysis of uterine preparations from ERKO females showed no detectable ER protein. Heterozygotes had one half the level of ER protein compared to wild-type animals. Estrogen insensitivity was confirmed using estrogen agonists, estradiol, hydroxy tamoxifen, diethylstilbestrol treatment for 3 days which resulted in a 3-4-fold increase in uterine wet weight and vaginal cornification in wild-type females, while ERKO mice were totally unresponsive. These data were further supported by the failure of estrogen or EGF treatment to induce DNA synthesis in uterine tissue of similarly treated mice. Lactoferrin, an estrogen-responsive gene in the uterus, was also assayed by Northern blot. Wild-type mice treated with a single estradiol injection showed a 350-fold induction in lactoferrin mRNA. while ERKO females showed no detectable response. Both male and female animals survive to adulthood with normal gross external phenotypes. As expected, females are infertile and demonstrate hypoplastic uteri and hyperemic ovaries with no apparent corpora lutea. Males are also infertile, with atrophy of the testes and seminiferous tubule dysmorphogenesis. Although the reproductive capabilities have been altered with a dramatic effect on the gonads, prenatal development of the reproductive tracts of both sexes appear to be independent of an ER-mediated response. Analysis of the mammary glands of the ERKO females at 4 months of age showed a primitive ductal rudiment rather than the fully developed ductal tree seen in wild-type siblings. Also absent were the terminal end buds seen during normal ductal morphogenesis. Both sexes show a decrease in skeletal bone density, supporting a direct role for ER action in bone. A single patient is described who is homozygous for a point mutation in the human ER gene at codon 157. The mutation produces a truncation of the ER protein and results in estrogen insensitivity syndrome. Most significant of the clinical findings are effects on skeletal bone density and retarded bone age. Findings from the patient and mice suggest that the absence of functional ER is not lethal. Mutation in the ER gene is present in the human population. Further characterization of the mice and identification of additional patients will be required to more fully understand the consequences of ER gene mutations.

Amino Acid Sequence↗