Alkaline phosphatase: a marker of alveolar type II cell differentiation

In an effort to identify type II cells by a method independent of staining phospholipid inclusions, we evaluated a histochemical technique for alkaline phosphatase activity in normal rat lung, in freshly isolated type II cells, and in primary culture of type II cells. In the adult rat alveolus, alkaline phosphatase staining selectively identified type II cells, although nonciliated bronchiolar (Clara) cells and loose perivascular connective tissue also stained for alkaline phosphatase activity. In cell suspensions of type II cells and other dissociated lung cells, alkaline phosphatase staining correlated closely with the modified Papanicolaou technique and was particularly useful in distinguishing type II cells from alveolar macrophages. To determine if alkaline phosphatase was related to the differentiated phenotype of type II cells, we studied conditions known to affect other type II cell functions. When type II cells were cultured on plastic substrata, the intensity of alkaline phosphatase staining decreased with increasing time in culture. To quantitate the apparent decrease in alkaline phosphatase activity, we used a biochemical assay to study the expression of alkaline phosphatase by type II cells. The specific activity of alkaline phosphatase in type II cells declined with increasing time in tissue culture on plastic substrata. Alkaline phosphatase activity was maintained, however, by culturing cells on Englebreth-Holm-Swarm (EHS) tumor matrix. Cells that had reduced levels of alkaline phosphatase activity following 48 h of culture on plastic substrata could be "rescued" by removing them from the plastic substratum and reculturing them for 48 h on EHS matrix. Alkaline phosphatase activity was also increased by culturing type II cells in the presence of cAMP or sodium butyrate.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of substratum and serum on the lipid synthesis and morphology of alveolar type II cells in vitro

To determine the effect of various culture conditions on the maintenance of lipid synthesis and morphology in alveolar type II cells, we cultured isolated adult rat alveolar type II cells on either plastic or denuded human amnionic basement membrane (ABM) in medium supplemented with either fetal bovine, porcine, horse, rat, or human serum. Lipid synthesis was assessed by incubation with [1-14C]acetate and determination of the distribution of radiolabel into individual lipid classes. Cells cultured on ABM incorporated significantly higher percentages of acetate into either phosphatidylcholine (PC) or phosphatidylglycerol (PG), and retained lamellar inclusions and a more characteristic cuboidal shape for longer periods than did cells cultured on plastic. Compared to other sera, cells cultured in the presence of rat serum incorporated the highest percentages of acetate into PC and saturated PC, had the best preservation of lamellar-body ultrastructure, and also appeared to contain more multivesicular bodies. The percent composition of linoleic acid, an essential fatty acid, was found to vary widely among the different sera. Supplementing media with linoleic acid resulted in a marked increase in acetate incorporation into saturated PC and a decreased incorporation into PG. We conclude that for maintenance of differentiated function of adult rat alveolar type II cells in primary culture (1) ABM is preferable to plastic as a culture substratum, (2) rat serum is preferable to fetal bovine serum as a serum supplement, and (3) the regulation of lipid synthesis by linoleic acid causes disparate effects on PG and saturated PC synthesis.     

Cell-cell and cell-matrix interactions differentially regulate the expression of hepatic and cytoskeletal genes in primary cultures of rat hepatocytes.

Freshly isolated adult rat hepatocytes exhibit a flat, extended morphology when cultured on dried rat tail collagen in the presence of growth factors; they actively synthesize DNA and express high levels of cytoskeletal mRNAs and proteins (actin, tubulin, cytokeratins, vinculin, alpha-actinin, and desmoplakin), while exhibiting low levels of liver-specific mRNAs (albumin, alpha 1-inhibitor III, and alpha 1-antitrypsin) and limited synthesis and secretion of albumin. Hepatocytes cultured on hydrated gel matrix from the Engelbreth-Holm-Swarm (EHS) mouse tumor form small spherical aggregates and exhibit low DNA, cytoskeletal mRNA, and protein synthesis, while at the same time exhibiting elevated liver-specific mRNAs and albumin production; these cells, therefore, more nearly conform to the program of gene expression seen within the normal animal. Hepatocytes on hydrated rat tail collagen resemble those on dry collagen when cultured at low density, but at high density they form compact trabecular aggregates, synthesize negligible amounts of DNA, and maintain a pattern of gene expression resembling that of hepatocytes seeded on the EHS matrix. If cell morphology is compact, as on EHS or on hydrated rat tail collagen when densely populated, DNA synthesis and expression of cytoskeletal genes are low, while liver-specific mRNAs are abundant. When cells are extended the opposite is the case. Without the growth supplement DNA synthesis is low throughout but gene expression is little affected. These studies point to the importance of cell-cell and cell-matrix interactions in determining the differentiated phenotype of hepatocytes, and they reveal an inverse relationship between cytoskeletal and liver-specific protein expression.     

Actin organization and hepatocyte differentiation are regulated by extracellular matrix via PI-4,5-bisphosphate in the rat

Cell adhesion to the extracellular matrix (ECM) plays vital roles in both morphogenesis and regulation of gene expression in cells of adult organisms. How intracellular, cytoskeletal, and signaling factors connect and communicate with the ECM is a fundamental question. Using a cDNA microarray analysis, we identified phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2) phosphatase mRNA as being up-regulated in hepatocytes cultured on a basement membrane matrix, Engelbreth-Holm-Swarm (EHS) gel, which led to the finding that the PI(4,5)P2 levels of hepatocytes decreased on EHS gel. These changes in hepatocytes on EHS gel were accompanied by promotion of actin depolymerization and differentiated phenotypes of the hepatocytes. Treatment with PI(4,5)P2 or a phospholipase C inhibitor, U73122, resulted in decreased mRNA expressions of albumin and hepatocyte nuclear factor 4 (HNF-4) in hepatocytes. In contrast, actin-disrupting agent gelsolin increased mRNA expressions of albumin and HNF-4. In conclusion, organization of the actin cytoskeleton via PI(4,5)P2 is involved in the regulation of hepatocyte differentiation by the ECM.     

Inhibitory effect of alcohol on osteogenic differentiation in human bone marrow-derived mesenchymal stem cells

BACKGROUND: Alcohol-induced osteoporosis is characterized by a considerable suppression of osteogenesis. The objective of this investigation was to determine the effect of alcohol on gene expression, protein synthesis, and mineralization in human bone marrow-derived mesenchymal stem cells induced toward osteogenic differentiation in vitro. METHODS: Human bone marrow-derived mesenchymal stem cells induced toward osteogenesis were cultured in the presence or absence of 50 mM alcohol. Stem cells were characterized by using SH2 antibody to the cell-surface antigen CD105/endoglin, and their proliferation in the presence of alcohol was quantified. The expression of genes for early, middle, and late markers of the osteogenic lineage was quantified by Northern analysis, and bone matrix protein synthesis was assayed. The effect of alcohol on cell-mediated matrix mineralization in terminally differentiated cultures was determined by von Kossa staining. RESULTS: Fluorescence-activated cell sorting analysis of human mesenchymal stem cells separated with a Percoll gradient proved 99% homogeneity by using SH2 antibody to the surface antigen CD105. Dose-dependent inhibition of proliferation of these stem cells occurred at concentrations greater than 50 mM alcohol. Gene expression of osteoblast-specific factor 2/core binding factor a1 (Osf2/Cbfa1), type I collagen, alkaline phosphatase, and osteocalcin (early, middle, and late markers for osteogenesis, respectively) was analyzed with and without osteogenic induction and treatment with 50 mM alcohol. After induction, Osf2/Cbfa1 levels were unresponsive to alcohol. To determine the effect of alcohol on human mesenchymal stem cell progression along the osteogenic pathway, messenger RNA (mRNA) levels for type I collagen, alkaline phosphatase, and osteocalcin were examined after osteogenic induction. After osteogenic induction, alcohol down-regulated the gene expression of type I collagen and significantly reduced its synthesis. Alcohol did not alter mRNA expression of alkaline phosphatase, a midstage marker for osteogenesis, but significantly decreased its activity compared with osteogenic induction alone. After induction, osteocalcin remained unchanged by alcohol at both the mRNA and protein levels. Histochemistry revealed decreased alkaline phosphatase staining and fewer alkaline phosphatase-positive cells in alcohol-treated human mesenchymal stem cell cultures. von Kossa staining revealed a reduction in the number of mineralizing nodules in stem cell cultures after alcohol treatment. CONCLUSIONS: Collectively, the data suggest that alcohol alters osteogenic differentiation in human bone marrow-derived mesenchymal stem cell cultures during lineage progression and provide further insight into alcohol-induced reduced bone formation.     

Influence of a reconstituted basement membrane and its components on casein gene expression and secretion in mouse mammary epithelial cells.

When primary mouse mammary epithelial cells are cultured on plastic, they rapidly lose their ability to synthesize and secrete most milk proteins even in the presence of lactogenic hormones, whereas cells cultured on released type I collagen gels show greatly enhanced mRNA levels and secretion rates of beta-casein and of some other milk proteins. We show here that culture on a reconstituted basement membrane from Engelbreth-Holm-Swarm tumor (EHS) allows greater than 90% of cells to produce high levels of beta-casein. By comparison, 30-40% of cells on released type 1 gels and only 2-10% of cells on plastic express beta-casein after 6 days in culture. Because only 40% of cells from late pregnant gland produced beta-casein before culture, the EHS matrix can both induce and maintain an increased level of casein gene expression. Individual basal lamina components were also evaluated. Type IV collagen and fibronectin had little effect on morphology and beta-casein mRNA levels. In contrast, both laminin and heparan sulfate proteoglycan increased beta-casein mRNA levels (1.5- to 4-fold and 2- to 8-fold, respectively). However, for heparan sulfate proteoglycan, increased message was not accompanied by increased secretion of beta-casein. Profound morphological differences were evident between cells cultured on plastic and on EHS matrix, the latter cells forming ducts, ductules, and lumina and resembling secretory alveoli. These results emphasize the vital role of the extracellular matrix in receiving and integrating structural and functional signals that can direct specific gene expression in differentiated tissues.     

Human fetal enterocytes in vitro: modulation of the phenotype by extracellular matrix.

The differentiation of small intestinal epithelial cells may require stimulation by microenvironmental factors in vivo. In this study, the effects of mesenchymal and luminal elements in nonmalignant epithelia] cells isolated from the human fetus were studied in vitro. Enterocytes from the human fetus were cultured and microenvironmental factors were added in stages, each stage more closely approximating the microenvironment in vivo. Four stages were examined: epithelial cells derived on plastic from intestinal culture and grown as a cell clone, the same cells grown on connective tissue support, primary epithelial explants grown on fibroblasts with a laminin base, and primary epithelial explants grown on fibroblasts and laminin with n-butyrate added to the incubation medium. The epithelial cell clone dedifferentiated when grown on plastic; however, the cells expressed cytokeratins and villin as evidence of their epithelial cell origin. Human connective tissue matrix from Engelbreth-Holm-Swarm sarcoma cells (Matrigel) modulated their phenotype: alkaline phosphatase activity increased, microvilli developed on their apical surface, and the profile of insulin-like growth factor binding proteins resembled that secreted by differentiated enterocytes. Epithelial cells taken directly from the human fetus as primary cultures and grown as explants on fibroblasts and laminin expressed greater specific enzyme activities in brush border membrane fractions than the cell clone. These activities were enhanced by the luminal molecule sodium butyrate. Thus the sequential addition of connective tissue and luminal molecules to nonmalignant epithelia] cells in vitro induces a spectrum of changes in the epithelial cell phenotype toward full differentiation.     

Characterization of the phosphatidylinositol-glycan membrane anchor of human placental alkaline phosphatase.

Placental alkaline phosphatase [orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] is a member of a diverse group of membrane proteins whose attachment to the lipid bilayer is mediated by a phosphatidylinositol-glycan. To investigate structural aspects of the glycolipid anchor, cultured WISH cells were used because we found that they produce the enzyme in abundant quantities. When cell suspensions were incubated with purified phosphatidylinositol-specific phospholipase C, most of the placental alkaline phosphatase was released from membranes in a hydrophilic form. On incubation of the cells with [14C]ethanolamine, [14C]myristic acid, or myo-[3H]inositol, each was incorporated into the phosphatase near the carboxyl terminus, showing that these components, which are found in other phosphatidylinositol membrane-linked proteins, are also present in placental alkaline phosphatase.     

Effects of tumor necrosis factor on bone formation in vitro

Tumor necrosis factor (TNF) was studied for its effects on bone formation in cultured rat calvariae. TNF alpha at 100-100,000 U/ml stimulated [3H]thymidine incorporation into DNA, an effect that appeared after 24 h of treatment and lasted 96 h. Transient (24-h) treatment with TNF alpha increased [3H]proline incorporation into type I collagen 24-72 h after the factor was removed; this effect was DNA synthesis dependent and blocked by hydroxyurea. Transient treatment with TNF alpha also increased alkaline phosphatase activity. In contrast, continuous treatment with TNF alpha for 48-96 h caused a marked inhibition on [3H]proline incorporation into type I collagen and alkaline phosphatase activity. TNF alpha caused a small increase in collagen degradation. Lymphotoxin had similar effects to those of TNF alpha. In conclusion, TNF alpha stimulates calvarial DNA synthesis which causes an increased number of collagen-synthesizing cells, but TNF alpha has a direct inhibitory effect on osteoblastic function.     

Effect of arterial proteoglycans on the interaction of LDL with human monocyte-derived macrophages

The effect of human arterial proteoglycans (PG1) on the interaction of low density lipoprotein (LDL) with cultured human monocyte-derived macrophages (HMDM) was studied. LDL was insolubilized by treatment with chondroitin-6-sulfate-rich, partially purified PG1. The LDL, resolubilized in culture medium, was added to HMDM. The PG1 pretreated LDL induced lipid accumulation in the HMDM, converting them into foam cells. Mass determination of lipids by spectrophotometric and chromatographic procedures showed a 2-4-fold accumulation of triglycerides, phospholipids, unesterified cholesterol and cholesterol esters in 48 h, in the HMDM incubated with PG pretreated LDL, when compared to those incubated with native LDL. Incorporation of [14C]oleic acid into the HMDM lipid esters correlated with the accumulation. Association of 125I-labeled LDL and of fluorescent labeled LDL (3,3-octadecyl indocarbocyanine) to HMDM also indicated that the PG1-pretreatment of LDL increased its uptake. Density gradient centrifugation, isoelectric focusing and electron microscopy showed that, when added to the cells, the PG1 pretreated LDL was not aggregated or altered in its surface charge. However, controlled trypsin treatment and polypeptide pattern analysis indicate that the accessibility of apoB has been altered. The results suggest that changes in the surface of LDL, induced by the arterial PG1, lead to increased endocytosis of the lipoprotein and stimulation of lipid synthesis in the macrophages. The possibility that a similar process may cause lipid accumulation in arterial macrophages is discussed.     

Human synovium produces substances that inhibit DNA and stimulate proteoglycan and collagen synthesis by cultured human articular chondrocytes and synovial fibroblasts

The effects of synovial conditioned medium (SCM) on DNA, proteoglycan (PG), and protein-collagen synthesis and respective gene expressions, in human articular chondrocytes (AC) and DNA synthesis in synovial fibroblasts (SFb), were studied in monolayer culture. All SCM exhibited concentration-dependent inhibition of [3H]thymidine incorporation in both AC and SFb. In contrast, SCM from three OA patients stimulated [35S]SO4 and [3H]glycine incorporations and the expression (RT-PCR) of aggrecan- and type II collagen-specific mRNAs in AC. The production of agents that inhibit DNA synthesis was blocked by indomethacin and dexamethasone and stimulated by IL-1 beta and TNF-alpha. The inhibitory substances were not produced by heat-inactivated tissue nor cultured SFb or AC and were completely solubles in methanol. It is postulated that synovial tissue secretes lipids, most probably arachidonic acid metabolites. These may counteract growth of an inflammatory synovial pannus by inhibiting SFb proliferation and enhance repair of damaged tissues by stimulating the matrix synthesis.     

The STRO-1+ fraction of adult human bone marrow contains the osteogenic precursors

The monoclonal antibody STRO-1 identifies clonogenic bone marrow stromal cell progenitors (fibroblast colony-forming units [CFU-F]) in adult human bone marrow. These STRO-1+ CFU-F have previously been shown to give rise to cells with the phenotype of fibroblasts, adipocytes, and smooth muscle cells. In this study, the osteogenic potential of CFU- F derived from the STRO-1+ fraction of adult human bone marrow was determined. CFU-F were isolated from normal bone marrow aspirates by fluorescence activated cell sorting, based on their expression of the STRO-1 antigen. Osteogenic differentiation was assessed by the induction of alkaline phosphatase expression, by the formation of a mineralized matrix (hydroxyapatite), and by the production of the bone- specific protein osteocalcin. STRO-1+ cells were cultured in the presence of dexamethasone (DEX; 10(-8) mol/L), ascorbic acid 2- phosphate (ASC-2P; 100 mumol/L), and inorganic phosphate (PO4i; 2.9 mmol/L). After 2 weeks of culture, greater than 90% of the cells in each CFU-F colony stained positive for alkaline phosphatase using a monoclonal antibody specific for bone and liver alkaline phosphatase. Alkaline phosphatase activity was confirmed by histochemistry. A mineralized matrix developed in the CFU-F cultures, after 4 weeks of culture in the presence of DEX, ASC-2P, and PO4i. Mineralization was confirmed by both light and electron microscopy. The mineral was identified as hydroxyapatite by electron dispersive x-ray microanalysis and by x-ray diffraction analysis. In replicate cultures, osteocalcin release was shown after exposure of the cells to 1,25-dihydroxyvitamin D3 (10(-7) mol/L) both by radioimmunoassay and Northern blot analysis. This work provides direct evidence that adult human bone marrow-derived CFU-F are capable of differentiating into functional osteoblasts and that osteoprogenitors are present in the STRO-1+ population.     

Vectorial secretion of prostaglandins by polarized rodent uterine epithelial cells

Uterine epithelial cells (UEC) isolated from mature mice as well as immature mice and rats were cultured on EHS matrix-coated nitrocellulose filters in order to determine their ability to secrete prostaglandin (PG) F2 alpha and PGE2 in a polarized manner. Ultrastructural analyses were performed to validate the polar nature of mouse UEC and demonstrate the presence of separate apical and basolateral plasma membrane domains. These properties included the presence of tightly juxtaposed lateral membranes, apical microvilli, and a relatively flat basal surface. Biochemical indices of polarity included the preferential (approximately 5:1) basal uptake of [35S]methionine as well as a preferential (approximately 9:1) apical secretion of protein. UEC isolated from mice during the estrous and diestrous stages of the estrous cycle did not differ in their degree of polarity, as measured by these morphological and biochemical indices. UEC of estrous and diestrous mice as well as immature mice and rats preferentially secreted PGF2 alpha to the basal medium to an approximately 4-fold greater extent than to the apical medium. PGE2 was secreted at least 10-fold less than PGF2 alpha, and a preferential basal secretion could not be demonstrated. Polarized UEC accumulated relatively large cellular pools of PGF2 alpha, while nonpolarized cells grown on matrix-coated plastic did not. This difference was reflected by the inability of an inhibitor of PG biosynthesis, indomethacin, to inhibit PGF2 alpha secretion by polarized cells during short (4-h) incubations. In contrast, this drug effectively inhibited secretion in nonpolarized cells or polarized cells incubated with indomethacin for longer (24-h) intervals. Therefore, cellular PGF2 alpha pools apparently support continued secretion of this lipid even when de novo synthesis is transiently inhibited. Preferential basal secretion of PGF2 alpha was due to the polar nature of UEC, since disruption of tight junctions with EGTA modified the basal to apical ratio of PGF2 alpha secretion to near unity. Sodium azide inhibited the secretion of PGF2 alpha, indicating that PGF2 alpha secretion was energy dependent. PGF2 alpha secretion was not coupled to protein synthesis or secretion, since cycloheximide did not inhibit this process in polarized or nonpolarized cells. These studies describe the first evidence for polarized secretion of lipid-derived hormones by epithelial cells. The preferential basal secretion of PGF2 alpha may play an important role in regulating UEC interactions with the underlying stroma.     

Effects of short chain fatty acids on a new human colon carcinoma cell line (LIM1215).

The effects of short chain fatty acids on a colon carcinoma cell line, LIM1215, have been studied. Of the four short chain fatty acids tested only butyrate at 1 mmol/l and 10 mmol/l and acetate at 10 mmol/l had significant effects on this cell line. The addition of butyrate to growth medium affected the growth rate and the production of alkaline phosphatase, dipeptidyl peptidase IV and carcinoembryonic antigen. Butyrate at a final concentration of 1 mmol/l increased the doubling time of the cells from 26 hours to 72 hours and decreased the cloning efficiency of the cells from 1.1% to 0.054%. Alkaline phosphatase concentrations increased rapidly in cells cultured in 1 mmol/l butyrate reaching peak levels after four days with alkaline phosphatase concentrations increasing more than six-fold. Levels of dipeptidyl peptidase IV and carcinoembryonic antigen were also increased after culture in butyrate containing medium. The number of alkaline phosphatase containing and dipeptidyl peptidase IV containing cells increased markedly in butyrate containing cultures. In contrast the number of mucus containing cells decreased in cultures grown in medium containing butyrate. This differentiating effect of butyrate on colon carcinoma cells may be relevant to the presence of butyrate in the colonic contents and the relationship between short chain fatty acids and fibre intake.     

A study of capillary pericyte viability on extracellular matrix produced by endothelial cells in high glucose

AIMS/HYPOTHESIS: Thickening of the basement membrane and selective loss of pericytes occur early in diabetic retinopathy. As we showed previously that pericyte adhesion is impaired on extracellular matrix produced by endothelial cells in high hexose concentrations, we aimed to verify if altered adhesion could influence pericyte viability and replication. METHODS: Conditioned extracellular matrices were obtained by growing human umbilical vein endothelial cells in media containing 28 mmol/l D-glucose, with or without the inhibitors of protein glycation thiamine or aminoguanidine, and D-galactose or L-glucose up to 28 mmol/l. Having removed the endothelium, bovine retinal pericytes were grown on these matrices and, in separate experiments, on laminin, fibronectin or type IV collagen. Pericyte viability and replication were measured by cell counts and DNA synthesis after 7 days, cell cycle traversal after 2 days and apoptosis after 18 h, 2 days and 7 days. RESULTS: Pericyte counts and DNA synthesis were reduced on matrices produced in high D-glucose and D-galactose, whilst matrix obtained in L-glucose reduced DNA synthesis but not counts. Both thiamine and aminoguanidine corrected reduced pericyte viability when added to high D-glucose. Cell cycle and apoptosis were not affected by growing pericytes on different conditioned matrices. Laminin, fibronectin and type IV collagen did not modify pericyte replication. CONCLUSIONS/INTERPRETATIONS: Reduced pericyte counts could depend on impaired initial adhesion to the extracellular matrix produced by endothelium in high hexose concentrations, rather than impaired replication or viability. Altered cell-matrix interactions might facilitate pericyte dropout in diabetic retinopathy, independently of the effects of high glucose on pericyte replication.     

Actions of recombinant human gamma-interferon and tumor necrosis factor alpha on the proliferation and osteoblastic characteristics of human trabecular bone cells in vitro

Using cultured human osteoblast-like cells, we studied the effects of tumor necrosis factor (TNF) and recombinant human gamma-interferon (gamma-IFN) on osteoblast growth and function, and demonstrated that TNF stimulated bone cell proliferation and prostaglandin production while inhibiting 1,25-(OH)2D3-stimulated alkaline phosphatase activity and osteocalcin release. In contrast, gamma-IFN inhibited proliferation and stimulated alkaline phosphatase activity of the cells, while inhibiting 1,25-(OH)2D3-stimulated osteocalcin production and having variable effects on the release of prostaglandins, depending on the presence of other factors. Our results suggest that TNF and gamma-IFN can act directly on bone-forming cells to affect both their proliferation and their differentiated function, and that changes in the ability of cells to produce these factors in disease states may contribute to alterations in the integrity of connective tissue matrices.     

Effects of triiodothyronine (T3) and identification of specific nuclear T3-binding sites in cultured human fetal epiphyseal chondrocytes.

The effects of T3 on cultured human fetal epiphyseal chondrocytes were assessed by studying its effects on DNA synthesis and alkaline phosphatase activity. DNA synthesis was evaluated as follows: after 48-h incubation in Ham's F-12 serum-free medium, cultured chondrocytes were incubated with or without T3 (0.1-100 nM) in MCDB-104 serum-free medium for different periods of time (2-10 days), with the addition of [3H]thymidine (5 microCi/mL) for the last 24 h. Confluent cultured chondrocytes in 25-cm2 tissue culture flasks were incubated in Ham's F-12 serum-free medium for up to 9 days with or without T3 (0.1-100 nM); the cellular cytoplasmic fraction was obtained, and alkaline phosphatase activity was evaluated using paranitrophenylphosphate as a substrate. No significant effects of T3 (0.1-100 nM) on DNA-[3H]thymidine incorporation were observed in any experiment (n = 17) for any gestational age (12-39 weeks) or for any incubation period studied (2-10 days). However, a significant (P less than 0.025 or more) stimulatory effect of T3 (0.1-100 nM) on alkaline phosphatase activity was observed after 9 days of incubation. This effect was highest for 5 nM T3 and was present in cultured chondrocytes from human fetuses of all ages studied (13-40 weeks). Cultured human fetal epiphyseal chondrocytes from human fetuses 12-40 weeks old (n = 8) showed specific nuclear binding sites for T3. The binding capacity was 27.14 +/- 2.84 fmol/100 micrograms DNA, and the Kd was 0.66 +/- 0.14 x 0.1 nM (mean +/- SEM), with no significant differences among fetal ages. In conclusion, our results show that T3 elicits a biological response in cultured human fetal epiphyseal chondrocytes and has specific nuclear binding sites. Since alkaline phosphatase is closely related to the mineralization of epiphyseal cartilage, these results suggest that thyroid hormones could regulate this process.     

Parathyroid hormone stimulates the proliferation of cells derived from human bone

Despite its acute inhibitory effect on bone formation in vitro, PTH has been shown to have an anabolic effect on bone in vivo and to stimulate cell proliferation in osteoblastic cell lines and organ cultures. We have examined the effects of PTH on cells derived from human trabecular bone and compared these effects with those on human skin fibroblasts. Human bone cells have the capacity to synthesize type I collagen and osteocalcin, and to respond to 1,25-dihydroxyvitamin D3 with an increase in the synthesis of osteocalcin and alkaline phosphatase. PTH stimulated adenylate cyclase activity at both low and high cell density. However, the same concentrations of hormone stimulated the proliferation of these cells only when they were cultured at a high cell density. The effect of PTH was bone cell specific in that no proliferative effect of PTH was detected in cultures of human skin fibroblasts obtained from the same donor and cultured under the same conditions. The effect of PTH on DNA synthesis by human bone cells may be important in the generation of a long term anabolic response to PTH.     

Thrombospondin functions as a cytoadhesion molecule for human hematopoietic progenitor cells

We explored the role that thrombospondin (TSP), a multifunctional extracellular matrix protein, plays in hematopoietic cell-cell and cell-matrix interactions. Thrombospondin synthesis is differentially regulated in human long-term bone marrow cultures. Consistent with this, human hematopoietic progenitor cells of all three lineages (erythrocyte, megakaryocyte, and granulocyte) use TSP as an attachment protein. However, terminally differentiated cells (erythrocytes and neutrophils) show absent or reduced attachment to TSP. The region within the TSP molecule that mediates cell attachment (cell binding domain) was delineated by examining both attachment to proteolytic fragments of TSP and by inhibition of cytoadhesion using monoclonal antibodies directed against TSP domains. The cell binding domain resides toward the C-terminus of a 140 Kd chymotryptic fragment of TSP. We conclude that thrombospondin functions as a hematopoietic cytoadhesion molecule, capable of binding primary hematopoietic progenitor cells, and may, therefore, be important in blood cell development.