Mechanisms and consequences of activation of protein kinase B/Akt

Chondrocytes isolated from the caudal and cephalic ends of the sterna of embryonic chicks were cultured in collagen gels. Differences were found, histochemically, between the proteoglycans produced by the "caudal" and "cephalic" cultures and with length of time in culture. The cultures were labelled with [14C]galactose and [35S]sulphate at 7 and 21 days in culture and labelled compounds from media, and cell and matrix extracts analysed with Sepharose CL-2B. A large aggrecan-like proteoglycan was detected in the media with some aggregated proteoglycans found in the cell extracts even under the dissociating conditions used. One group of 14C-labelled compounds, found in the cell and matrix extracts, was equivalent in size to chick aggrecan core protein. Smaller proteoglycans and glycoprotein glycans were present. The types and proportions of these proteoglycans varied between the two cell types demonstrating biosynthetic commitment.     

The role of 12-lipoxygenase in pancreatic -cells (Review)

A culture system that facilitates the chondrogenic differentiation of rabbit bone marrow-derived mesenchymal progenitor cells has been developed. Cells obtained in bone marrow aspirates were first isolated by monolayer culture and then transferred into tubes and allowed to form three-dimensional aggregates in a chemically defined medium. The inclusion of 10(-7) M dexamethasone in the medium induced chondrogenic differentiation of cells within the aggregate as evidenced by the appearance of toluidine blue metachromasia and the immunohistochemical detection of type II collagen as early as 7 days after beginning three-dimensional culture. After 21 days, the matrix of the entire aggregate contained type II collagen. By 14 days of culture, there was also evidence for type X collagen present in the matrix and the cells morphologically resembled hypertrophic chondrocytes. However, chondrogenic differentiation was achieved in only approximately 25% of the marrow cell preparations used. In contrast, with the addition of transforming growth factor-beta 1 (TGF-beta 1), chondrogenesis was induced in all marrow cell preparations, with or without the presence of 10(-7) M dexamethasone. The induction of chondrogenesis was accompanied by an increase in the alkaline phosphatase activity of the aggregated cells. The results of RT-PCR experiments indicated that both type IIA and IIB collagen mRNAs were detected by 7 days postaggregation as was mRNA for type X collagen. Conversely, the expression of the type I collagen mRNA was detected in the preaggregate cells but was no longer detectable at 7 days after aggregation. These results provide histological, immunohistochemical, and molecular evidence for the in vitro chondrogenic differentiation of adult mammalian progenitor cells derived from bone marrow.     

Metabolism of the extracellular matrix formed by intervertebral disc cells cultured in alginate

STUDY DESIGN: Cells from normal rabbit nucleus pulposus (NP) and anulus fibrosus (AF) were cultured in alginate beads for as long as 14 days to allow them to reform a matrix made up of two compartments: the cell-associated matrix (CM) and further removed matrix (FRM). At different time points, the CM and FRM made by each cell population were analyzed using histologic, biochemical, and immunologic assays. OBJECTIVES: To study the metabolism of normal rabbit NP and AF cells in alginate by characterizing the CM and FRM formed by each cell population, and to identify metabolic properties that may shed light on mechanisms at play in disc degeneration. SUMMARY OF BACKGROUND DATA: Little is known about the metabolism of intervertebral disc cells, in part because of the lack of microculture systems appropriate for the study of these cells in vitro. In recent studies from our laboratories, it was suggested that articular chondrocytes cultured in alginate beads remain phenotypically stable and reform a matrix similar to the one they populate in vivo. This culture system appears ideally suited for the study of intervertebral cells available only in limited numbers. METHODS: Rabbit NP and AF cells released from the matrix by sequential enzyme digestion were encapsulated in alginate beads (20,000 cells/bead) and cultured for as long as 14 days. At selected time points, beads were solubilized with calcium chelating agents, and the CM and FRM were isolated. The rate of 35S-sulfate incorporation into proteoglycans, and the contents of various extracellular matrix molecules (total sulfated proteoglycans, antigenic keratan sulfate, hyaluronan, collagen, and pyridinium crosslinks) were measured. RESULTS: Both NP and AF cells remained phenotypically stable in the alginate gel throughout the culture period and reestablished a matrix composed of CM and FRM compartments. The two cell populations exhibited numerous differences in their metabolic activities in vitro. Nucleus pulposus cells synthesized fewer proteoglycan and collagen molecules and were less effective in incorporating these into the CM than AF cells. CONCLUSIONS: Intervertebral disc cells, especially NP cells, are extremely sluggish in reforming a CM, a protective shell rich in proteoglycans and collagen molecules. This may help explain why damage to the NP often is accompanied by progressive degeneration of the disc in vivo.     

Variation of multifunctional surface binding proteins--a virulence strategy for group A streptococci?

Procollagen and proteoglycan biosynthesis was defined in long-term culture of a human osteogenic sarcoma cell line, SAOS-2. An osteoblast phenotype was maintained by these cells up to 40 days post-confluent in the presence of ascorbic acid. Under these conditions, cells deposited around them an extensive collagenous matrix that was able to mineralize in the presence of an exogenous phosphate donor (beta-glycerophosphate). The collagenous matrix was comprised predominantly of collagen type I with significant amounts of collagen type V, and greater than 80% of the collagen in the matrix was involved in covalent crosslinkages. With increasing time in culture there was a decrease in the collagen synthetic rate, although the collagenous matrix was maintained. The proteoglycans synthesized by nonmineralizing and mineralizing cultures were purified after biosynthetic labeling with [35S]sulfate and [3H]glucosamine. Two major species were apparent: a large chondroitin sulfate proteoglycan (CSPG), and a small chondroitin sulfate proteoglycan, decorin. In nonmineralizing cultures, decorin partitioned equally between the cell layer and culture medium, whereas the large CSPG species partitioned exclusively into the cell layer-associated matrix. In the presence of extensive mineral deposition, greater than 90% of the newly synthesized proteoglycans were secreted into the medium. Northern blot hybridization indicated that SAOS-2 cells express mRNA encoding a range of bone proteins, including decorin, osteonectin, and bone sialoprotein.     

Osteogenesis imperfecta type II: microvascular changes in the CNS

Osteogenesis imperfecta, type II is a rare hereditary disease of connective tissue with abnormalities of type I collagen. It is invariably fatal in the neonatal period. We report 2 cases with abnormal cerebral cortical white matter consisting of abundant, perivenous microcalcifications, proliferated vascular endothelium and focal aggregates of germ cells. Histochemically, microcalcifications lie within nodules composed of a PAS-positive, carbohydrate-rich matrix. This matrix also stains with alcian blue suggestive of a proteoglycan component. Immunoperoxidase staining reveals that some of these nodules are surrounded by type I collagen. Adjacent vessels show endothelial proliferation associated with markedly redundant basement membranes as confirmed by reactivity with anti-type IV collagen. Although the cerebral cortex has a normal neuronal cytoarchitecture, the areas that appear externally dysplastic overlie nests of germ cells within the underlying white matter suggestive of impaired migration. These changes most likely occurred during the third trimester and may involve abnormal interactions of proteoglycans and the extracellular matrix with collagen.     

Human chondrocytes proliferate and produce matrix components in microcarrier suspension culture

Chondrocytes propagated in monolayer culture proliferate and change into 'fibroblastoid'-like cells. This change is characterized by a shift in production of collagen type II to I and from high- to low-molecular-weight proteoglycans. When propagated in three-dimensional culture, chondrocytes have limited ability to divide but re-express their original characteristics. The goal of the present study was to determine whether a microcarrier suspension culture system would support chondrocyte proliferation and phenotype expression. Our experiments indicate that a collagen type I microcarrier (cellagen) best supported chondrocyte proliferation and phenotype expression. Cells in cellagen microcarriers multiplied at least twentyfold within 2 weeks and had doubling times of 2 to 3 d. Viable and metabolically active cells were retrieved with ease. The harvested chondrocytes had no detectable staining for collagen type I and stained intensely for collagen type II. Our studies demonstrate that the microcarrier suspension culture system supports growth and enhances expression of the 'chondrocytic' phenotype. Attachment to a constrained surface and the fluid shear forces on the microcarriers during suspension culture may have helped chondrocytes to reacquire their rounded shape and produce cartilage matrix components.     

New insights into the cytodynamics of the hamster Harderian gland as provided by the bromodeoxyuridine-labelling method

The fourth week of postnatal life is a critical point in the development of the hamster Harderian gland. During this week, cells with large lipid vacuoles (type-II cells) appear in the male gland, marking a morphological sex difference that is notorious in adult animals. The origin and fate of type-II cells are controversial. To gain insight into the mechanisms by which type-II cells become a major cell type in the gland of adult male hamsters, bromodeoxyuridine (BrdU) labelling was used to assess the proliferative activity of both types of glandular cells in 28-day-old animals. To search for possible sex differences in the proliferative activity of this gland, female animals of the same age as the males were also studied. No difference was found in the overall labelling index (BrdU-labelled cells/100 cells) between males (1.8 +/- 0.1%) and females (1.5 +/- 0.1%). In the gland of the males, the specific labelling index of type-II cells (3.4 +/- 0.4%) was significantly higher than that of type-I cells (0.9 +/- 0.2%). Interestingly, the proportion of type-II cells present in the male glands at this age (36.6%) was significantly lower than that of type-I cells. Our results strongly suggest that the proliferation of type-II cells, rather than a continuous differentiation of these cells from preexisting type-I cells, is a major event in the achievement of the mature form of this gland. The results reported here counsel a reappraisal of current theories about the cytodynamics of the hamster Harderian gland.     

Characterization of Borrelia spp. isolated from the tick, Ixodes tanuki and small rodents in Japan

Rat pancreatic periacinar fibroblastoid cells (PFCs) appear to be involved in intralobular fibrosis and acinar cell regeneration. We isolated pancreatic acini of the rat, cultured the fibroblastoid cells, and characterized the cells morphologically and immunohistochemically. Isolated acini were seeded on culture dishes, and spindle-shaped cells migrated and proliferated. On Electronmicroscopic examination, microfilament bundles were seen, and the intracellular localization of vimentin, alpha-smooth muscle actin, and non-muscle myosin was identified immunohistochemically. These findings strongly suggest that the cells were myofibroblast-like. The PFCs were also demonstrated, immunohistochemically, to contain prolyl hydroxylase, type-I procollagen, type-III procollagen, type-IV collagen, fibronectin, and laminin. Stimulation by transforming growth factor beta 1 (TGF beta 1) increased intracellular immunoreactive prolyl hydroxylase and collagen synthesis in the PFCs. These findings indicate that PFCs proliferate in culture as myofibroblast-like cells and synthesize extracellular matrix components. It is possible that PFCs are involved in intralobular fibrosis in response to stimulation with TGF beta 1.     

Phenotypic and functional characterization in vitro of a multipotent epithelial cell present in the normal adult human breast

The developmental relationships between the different mammary epithelial cell lineages in the human mammary gland are not well defined. To characterize human breast epithelial cells (HBEC) with progenitor activity, we used flow cytometry and single cell sorting to analyze the distribution of cellular phenotypes in primary cultures of reduction mammoplasties and their associated ability to generate colonies in 2-dimensional (D) and 3-D (collagen gel) culture systems. This approach allowed two distinct types of HBEC progenitor populations to be distinguished on the basis of their differential expression of the MUC-1 glycoprotein, CALLA/CD10 and epithelial-specific antigen (ESA). The first type of progenitor, which is enriched in the MUC-1+/CAL-LA-/ESA+ subpopulation, generated colonies of tightly arranged cells in 2-D cultures and small alveolar-like colonies with a central lumen when cultured in a collagen matrix. The cells produced in the colonies and derived from these MUC-1+/CALLA-/ESA+ progenitors were found to express typical luminal epitopes (keratin 8/18, keratin 19, MUC-1, ESA) and showed low levels of expression of myoepithelial epitopes (keratin 14 and CD44v6). The second type of progenitor, which is present in the MUC-1-to +/-/CALLA +/- to +/ESA+ subpopulation, generated mixed colonies of both luminal and myoepithelial cells when seeded in 2-D and 3-D cultures. In 2-D cultures, the centrally located cells exhibited a luminal morphology and expressed ESA, but were heterogeneous in their expression of MUC-1. Radiating from the periphery of these ESA+ HBEC were highly refractile ESA- teardrop-shaped myoepithelial-like cells. When cultured in a collagen matrix, these bipotent progenitors generated large branched colonies composed of a heterogeneous population of cells, with some of the progeny cells expressing luminal epitopes (keratin 8/18, keratin 19 and MUC-1) and others expressing myoepithelial epitopes (keratin 14 and CD44v6). A third type of progenitor, which became apparent is passaged HBEC cultures and was enriched in the MUC-1-/CALLA+/ESA- subpopulation, was found to generate colonies of cells with an exclusively myoepithelial phenotype. These results provide definitive evidence for the existence of multilineage HBEC progenitors in normal adult human mammary tissue. The phenotypic profile of these cells suggest that these multilineage progenitors are a relatively undifferentiated cell since they express low levels of MUC-1 and that they have a luminal location within the mammary epithelium since they are ESA+. Furthermore, we suggest that the MUC-1+/CALLA-/ESA+ and the MUC-1- to +/-/CALLA +/- to +/ESA+ progenitors we have identified and characterized are candidate in vivo alveolar and ductal progenitors, respectively.     

Genetic and clinical features of sensorineural hearing loss associated with the 1555 mitochondrial mutation

This article describes the modulation, by extracellular collagen, of DNA and proteoglycan synthesis in articular chondrocytes stimulated with transforming growth factor-beta 1. Type-I and type-II collagen, heat-denatured type-II collagen, and bovine serum albumin were each incorporated into alginate in increasing concentrations. Bovine articular chondrocytes were isolated and were resuspended in the alginate, yielding alginate beads with final extracellular protein concentrations of 0-1.5% (wt/vol) for the collagens and 0-2.5% (wt/vol) for bovine serum albumin. Cultures of beads were maintained for 7 days in basal Dulbecco's modified Eagle medium or in medium supplemented with 10 ng/ml transforming growth factor-beta 1. Subsequently, the synthesis of DNA and proteoglycan was measured by radiolabel-incorporation methods with [35S]sulfate and [3H]thymidine, and the values were normalized to the DNA content. Transforming growth factor-beta 1 stimulated the synthesis of both DNA and proteoglycan in a bimodal fashion. The presence of extracellular type-II collagen increased the rate of DNA and proteoglycan synthesis in a dose-dependent fashion in cultures stimulated by transforming growth factor-beta 1, whereas heat-inactivated type-II collagen abrogated the effects observed with type-II collagen for synthesis of both DNA and proteoglycan. In contrast, the presence of extracellular type-I collagen caused a dose-dependent inhibition of synthesis of both DNA and proteoglycan in cultures stimulated with transforming growth factor-beta 1. Extracellular bovine serum albumin brought about a limited increase in synthesis rates, presumably by blocking nonspecific cytokine binding. These results suggest that type-II collagen has a specific role in chondrocyte regulation and serves to mediate the response of chondrocytes to transforming growth factor-beta 1.     

High-frequency oscillation and centroid frequency of diaphragm EMG during inspiratory loading

Exposure of progenitor cells with chondrogenic potential to recombinant human osteogenic protein-1 [rhOP-1, or bone morphogenetic protein-7 (BMP-7] may be of therapeutic interest in the regeneration of articular cartilage. Therefore, in this study, we examined the influence of rhOP-1 on cartilage formation by human perichondrium tissue containing progenitor cells with chondrogenic potential in vitro. Fragments of outer ear perichondrium tissue were embedded in clotting autologous blood to which rhOP-1 had been added or not (controls), and the resulting explant was cultured for 3 weeks without further addition of rhOP-1. Cartilage formation was monitored biochemically by measuring [³5;S]sulfate incorporation into proteoglycans and histologically by monitoring the presence of metachromatic matrix with cells in nests. The presence of rhOP-1 in the explant at the beginning of culture stimulated [³5;S]sulfate incorporation into proteoglycans in a dose-dependent manner after 3 weeks of culture. Maximal stimulation was reached at 40 microgram/ml. Histology revealed that explants treated with 20-200 microgram/ml rhOP-1, but not untreated control explants, contained areas of metachromatic-staining matrix with chondrocytes in cell nests. These results suggest that rhOP-1 stimulates differentiation of cartilage from perichondrium tissue. The direct actions of rhOP-1 on perichondrium cells to stimulate chondrocytic differentiation and production of cartilage matrix in vitro provide a cellular mechanism for the induction of cartilage formation by rhOP-1 in vivo. Thus, rhOP-1 may promote early steps in the cascade of events leading to cartilage formation. Therefore, rhOP-1 could be an interesting factor for regeneration of cartilage in articular cartilage defects.     

Anatomy and histological characteristics of the spinoglenoid ligament

The spinoglenoid (inferior transverse scapular) ligament, when present, is located at the spinoglenoid notch. The ligament originates on the spine of the scapula and inserts on the superior margin of the glenoid neck. Because of discrepancies in the literature, we sought to determine its prevalence and to define its histological characteristics. We dissected 112 shoulders of seventy-six cadavera and classified the ligament as absent or an insubstantial structure, a thin fibrous band (type I), or a distinct ligament (type II). We found no distinct ligamentous structure in twenty-two shoulders (20 percent), a type-I ligament in sixty-eight shoulders (61 percent), and a type-II ligament in twenty-two shoulders (20 percent). Overall, ninety (80 percent) of the shoulders had a fibrous band of tissue that, together with the spine of the scapula, formed a narrow fibro-osseous tunnel through which the suprascapular nerve traveled. The bone-spinoglenoid ligament-bone complexes from three specimens were analyzed histologically. There were two type-I ligaments and one type-II ligament; all three ligaments were composed of collagen fibers. One type-I ligament and the type-II ligament demonstrated Sharpey fibers at their origin on the spine of the scapula. The other type-I ligament attached to the spine of the scapula through the periosteum. All three ligaments inserted into the periosteum of the glenoid neck.     

Distribution of proteoglycans in the trabecular tissue of eyes with neovascular glaucoma

We investigated histo-chemically the composition and distribution of proteoglycans in the trabecular tissue of eyes with neovascular glaucoma. Cupromeronic blue in combination with a series of enzyme digestions and nitrous acid treatment were used. The spaces between the trabecular beams were lined by a single layer of vascular endothelium and were filled with red blood cells. A basal lamina and microfibrils were detected just beneath the newly formed vascular endothelial cells. Chondroitin-sulfate- and dermatan-sulfate-type proteoglycans were present in association with collagen fibrils in the extracellular matrix. Heparan-sulfate-type proteoglycans were present in association with the basal lamina of both the vascular endothelial cells and the trabecular cells. It is unlikely that these abnormalities in the type or distribution of proteoglycans in the trabecular meshwork have a major role in the pathogenesis of glaucoma.     

Diagnostic value of atypical lymphocytes in cerebrospinal fluid from adults with enteroviral meningitis

We have noted two morphologically distinct types of atypical lymphocytes (AL) in the cerebrospinal fluid (CSF) of adult patients with meningitis: one, which we designate type-I AL, with multilobulated nuclei resembling those of the abnormal cells in adult T-cell leukaemia (ATL); and another, type-II AL, characterized by large lymphocytes with basophilic cytoplasm and nuclei containing coarse chromatin. Type-I AL were detected in 25 of 39 patients (64%) with enteroviral and in 11 of 109 (11%) with aseptic meningitis presumed to be caused by other viruses, but not in meningitis resulting from Cryptococcus neofirmans (n = 14), Mycobacterium tuberculosis (n = 19) or acute bacterial infection (n = 49). Type-I AL were not seen in herpes zoster (n = 15) aseptic meningeal reactions (n = 15), or in leptomeningeal carcinomatosis (n = 14). Type-II AL were often present in meningitis of various aetiologies and in aseptic meningeal reactions, but not in leptomeningeal carcinomatosis. The presence of type-I AL in the CSF was found to be indicative of enteroviral meningitis with the highest predictive value (69%), while type-II AL had a lower diagnostic positive predictive value in meningitis of the five aetiologies above. Type-I AL immunostained for CD4, while type-II AL were stained for CD8. The presence of type-I AL in CSF strongly suggests enteroviral meningitis, which warrants careful follow-up without antifungal, antituberculous or antibacterial agents. However, type-I AL, which are likely to be virally transformed lymphocytes, must be distinguished from ATL cells, which frequently involve the meninges.     

Scanning electron microscopy of normoxic and hyperoxic hyperbaric exposed lungs

Lungs from adult guinea pigs exposed to 1 ATA He-O2 with 200 mm Hg PO2 and 20 ATA He-O2 with 200, 400, and 600 mm Hg PO2 were studied with scanning electron microscopy. The appearance of normal alveoli is described. Even before pulmonary O2 toxicity became symptomatic, subtle changes occurred in the alveoli, such as an increase in macrophages and a marked increase in length of alveolar type-II cell microvilli. These changes occurred in animals exposed to 400 mm Hg PO2, heretofore considered below toxic levels. With increased toxic involvement, the number of alveolar type-II cells increased. A thick layer of material appeared in some of the alveoli, obscuring the Kohns pores and type-I and -II cell surfaces. The alveolar-capillary network with underlying erythrocytes was no longer observable. Lungs with the greatest toxic involvement possessed large numbers of macrophages encompassed by a fibrin-like matrix. The alveolar walls were broken down in many instances, and the alveoli were no longer discrete units but took on the appearance of an amorphous mass of lung tissue.     

Genetic stability of oral polio vaccine prepared on primary monkey kidney cells or Vero cells--effects of passage in cell culture and the human gastrointestinal tract

The genetic stability of the three Sabin oral poliovaccine (OPV) strains produced on either primary monkey kidney (VK) or Vero cell substrates was compared in vivo and in vitro by measuring the rate at which the bases most strongly associated with attenuation and reversion to neurovirulence (positions 480, 481, and 472 in the 5' non-coding region of Sabin 1, 2 and 3 respectively, and 2034 in VP3 of Sabin 3) reverted during passage of the vaccine strains in the gastrointestinal tract of primary vaccinees and in cell culture. For the in vivo study, the poliovirus excretion patterns of 21 infants receiving OPV produced on either VK or Vero cells were followed for 21 days. No significant differences in either the frequency of excretion or the rate of reversion were observed between the two vaccine groups. The rate of accumulation of revertants during passage in vitro was compared for the three Sabin strains passaged 10 times in either VK or Vero cells. For types 1 and 3, revertants accumulated faster upon passage through VK cells compared with passage through Vero cells. Type 2 appeared to be stable as no revertants were detected in either cell type. Results of this study suggest that the use of Vero as opposed to VK cells as substrate for the manufacture of OPV does not negatively influence the genetic stability of the three Sabin OPV strains in vivo or in vitro.     

The synthesis of elastin, collagen, and glycosaminoglycans by high density primary cultures of neonatal rat aortic smooth muscle. An ultrastructural and biochemical study

Primary cultures of neonatal rat aortic smooth muscle cells inoculated at high densities (1 X 10(6) cells/25 cm2 Falcon flask) with adequate nutrient media and pH control grow rapidly and form multilayers of cells with typical "hill and valley" organization. After 10 days growth insoluble elastin formation could be visualized by phase contrast microscopy as small particles which grew rapidly to become larger irregular refractile aggregates and later coalesced to form larger aggregates and small fibres. With light and electronmicroscopy, elastin was the predominant matrix protein formed, with the "hill regions" of cultures containing abundant elastin aggregates and some collagen. In 2-week-old cultures differentiation could be observed within the cell multilayer. The older deeper cells contained more protein synthesis organelles and myofilaments and were in close association with large often coalescing elastin aggregates; compared to younger more superficial cells which contained more free polyribosomes less myofilaments, and were associated with fewer and small elastin aggregates. In older cultures this differentiation was not apparent; the cells contained many myofilaments, dense bodies, and lysosomes. Elastin aggregates and newly formed elastic fibres were abundant in the matrix. Quantitative analysis of insoluble elastin formation in the cell layer during the 4-week culture period indicated continuous biosynthesis and deposition which paralleled that of desmosine formation. Amino-acid analysis of a hot alkali insoluble residue (regarded as elastin) from 30-day-old cultures gave a profile identical with neonatal rat aortic elastin in vivo. Insoluble collagen formation in the cell layer tended to plateau after the log phase of growth was completed (10 days). Proteoglycans were found predominantly in the supernatant media. Glycosaminoglycan analysis revealed a profile of dermatan sulphate (32%), chondroitin 4-sulphate (43%), keratan and heparan sulphate (30%), with only a trace of hyaluronic acid. This study indicates that primary cultures of neonatal rat aortic smooth muscle cells remain differentiated in culture and have the unique capacity to continue to synthesize and deposit large amounts (mg) of insoluble elastin which aggregate and from elastic fibres in vitro.     

The effect of glucocorticoid on the synthesis of biglycan and decorin in human osteoblasts and bone marrow stromal cells

We have previously demonstrated that glucocorticoids induce differentiation of human bone marrow stromal cells (BMSC) into cells expressing mature osteoblast phenotype. As glucocorticoids have marked effects on extracellular matrix protein synthesis in bone, and because proteoglycans are important components of bone matrix and may condition the differentiation and biological activities of osteoblasts, we studied the effects of dexamethasone (Dex) on the synthesis of small proteoglycans [decorin (DCN) and biglycan (BGN)] in adult human BMSC and human osteoblasts (HOB). First passaged HOB and BMSC were treated with either ethanol or 10(-7) M Dex for 7 days. After treatment, the cells were metabolically labeled with either [35S]SO4 alone or [35S]SO4 and [3H]leucine together for 24 h. Conditioned media were collected, and cell layers were extracted with 4 M guanidine HCl. The extracts and conditioned media were subjected to gel filtration and ion exchange chromatography. Fractions containing radiolabeled proteoglycans were analyzed either directly or after immunoprecipitation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Dex treatment resulted in a dramatic increase in DCN and an associated decrease in BGN in both the conditioned medium and the cell layer of HOB cultures. In Dex-treated BMSC cultures, BGN was decreased in both the conditioned medium and cell layer, whereas DCN was stimulated in the majority of cultures. Northern blot analysis indicated that steady state messenger RNA (mRNA) concentration of DCN was increased by Dex in all of the HOB cultures and in seven of eight BMSC cultures analyzed. The steady state mRNA level of BGN was decreased by Dex in both HOB and BMSC cultures. The regulation of DCN and BGN mRNA by Dex in both HOB and BMSC (when responsive) was dose dependent. Time-course analysis indicated that as little as 1 day of treatment with Dex was sufficient to decrease BGN mRNA and increase DCN mRNA (when observed) levels in BMSC; the regulation spanned a 4-week interval, during which the extracellular matrix of BMSC was mineralized. The effect of Dex on the steady state mRNA levels of DCN and BGN in HOB was also apparent after 1 day of treatment. These accumulated results suggest that Dex modulates the synthesis of small proteoglycans in both human bone marrow stromal osteoprogenitor cells and mature osteoblasts.     

Effect of transforming growth factor-beta on proteoglycan synthesis by chondrocytes in relation to differentiation stage and the presence of pericellular matrix

The effects of transforming growth factor-beta (TGF-beta) on proteoglycan synthesis of chondrocytes are controversial. The hypothesis that the differential effect of TGF-beta is related to the differentiation stage of the chondrocytes is investigated in this study. Rabbit auricular chondrocytes were cultured in alginate. When seeded in alginate immediately after isolation, cells keep their cartilaginous phenotype. When cells are first cultured in monolayer, they lose their cartilaginous phenotype and become dedifferentiated. We used three different cell populations: (1) Differentiated cells (P0: immediately after isolation); (2) partially (de)differentiated cells (P1: after one passage in monolayer); (3) dedifferentiated cells (P4: after four passages in monolayer). Cells were characterized by morphology using electron microscopy, amount of proteoglycans using the Farndale assay and type of collagen produced using immunohistochemistry. The effects of addition of 10 ng/ml TGF-beta2 for 7 days to P0, P1 and P4 cells were compared. TGF-beta was added either directly from the start of the alginate culture, or after a preculture period of three weeks in alginate. The amount of proteoglycans was increased in all chondrocyte populations when TGF-beta was added immediately after seeding in alginate, indicating that the effect of TGF-beta on proteoglycan synthesis does not depend on the differentiation stage of cells. After preculture in alginate, stimulation of proteoglycan synthesis (as measured by amount of proteoglycans and 35S-sulfate incorporation) had vanished. This effect was independent of differentiation stage . A dose-response experiment with TGF-beta (1, 10, 50 ng/ml) confirmed this differentiation-stage-independent effect of TGF-beta on proteoglycan synthesis. Stimulation by TGF-beta can be retained after enzymatic digestion of the pericellular matrix and reseeding of the cells in alginate, indicating the importance of pericellular matrix for the effect of TGF-beta on matrix synthesis. Alkaline phosphatase (ALP) activity was largely inhibited by TGF-beta in P0 chondrocytes, either with or without preculture in alginate. After culturing in monolayer, ALP activity was not substantially changed by TGF-beta. This indicates that the effect of TGF-beta on ALP activity, in contrast to the effect on proteoglycan synthesis, does depend on the differentiation stage of the cells. Furthermore, the fact that ALP synthesis in P0 cells is still inhibited by TGF-beta after preculture indicates that these cells remain responsive to TGF-beta. This provides additional evidence for the importance of the pericellular matrix for regulation of the effect of TGF-beta on proteoglycan synthesis. The results indicate that, in pathological cartilage, matrix depletion might be the trigger for increased matrix synthesis in reaction to TGF-beta, suggesting an important role for TGF-beta in cartilage repair.