Glucosamine promotes chondrogenic phenotype in both chondrocytes and mesenchymal stem cells and inhibits MMP-13 expression and matrix degradation.

OBJECTIVES: Glucosamine (GlcN), a natural amino monosaccharide, is a constituent of glycosaminoglycans (GAGs) found in hyaline cartilage. GlcN salts constitute a new class of nutraceutical components with putative chondroprotective activity, which may target chondrocytes as well as chondroprogenitors cells, such as mesenchymal stem cells (MSCs), during cartilage turnover and repair. In the present study, we examined the effects of GlcN on chondrogenesis of human MSCs (hMSCs) and the phenotype of normal and osteoarthritic human articular chondrocytes, using an in vitro pellet culture model maintained in a defined medium. METHODS: hMSCs and normal and osteoarthritic human chondrocytes grown as pellet cultures, stimulated or not with interleukin-1beta (IL-1beta), were treated with varying doses of GlcN. Expression of cartilage matrix genes and cartilage degrading enzymes was determined by semiquantitative and quantitative real-time reverse transcription polymerase chain reaction (RT-PCR), and by histological staining of cartilage markers, as well as sulfated GAG (sGAG) analysis and Western blotting. RESULTS: Chondrocytes grown in the presence of serum for 11 days showed decreased expression of the cartilage matrix genes, collagen type II (collagen II) and aggrecan, as early as day 3, which was reversed with GlcN treatment by day 11. Both hMSCs and chondrocytes grown as pellet cultures in defined medium and treated with 100 microM GlcN exhibited enhanced expression of collagen II and aggrecan as well as increased content of sGAG, when compared to control untreated pellets. However, high doses of GlcN (10-20mM) were inhibitory. GlcN treatment partially blocked IL-1beta mediated downregulation of collagen II and aggrecan expression and inhibited expression of the matrix degrading enzyme, matrix metalloproteinase 13 (MMP-13), in both chondrocytes and hMSCs undergoing chondrogenesis. CONCLUSIONS: These observations suggest that GlcN treatment enhances hMSC chondrogenesis and maintains cartilage matrix gene expression in chondrocytes, which may account for some of the reported chondroprotective properties of GlcN on cartilage.

Estrogenic endocrine disruptive components interfere with calcium handling and differentiation of human trophoblast cells.

During development, calcium (Ca) is actively transported by placental trophoblasts to meet fetal nutritional and the skeletal mineralization needs. Maternal exposure to estrogenic pesticides, such as 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane (DDT) and methoxychlor (MTC), has been shown to result in reproductive disorders and/or abnormal fetal development. In this study, we have examined the effects of exposure of trophoblastic cells to MTC and DTT, in comparison to 17beta-estradiol (E2) and diethylstilbestrol (DES), to test the hypothesis that cellular Ca handling is a target for these endocrine disruptive components. Treatment with DDT, MTC, DES, or E2 increased cellular Ca uptake, and the expression of trophoblast-specific human Ca binding protein (HCaBP) was down-regulated by both MTC and DDT. Treatment with MTC, DDT, and DES inhibited cell proliferation, induced apoptosis, and suppressed expression of several trophoblast differentiation marker genes. These effects were reversed by overexpression of metallothionein IIa, a gene highly responsive to cadmium and other metals. These results strongly suggest that trophoblast Ca handling functions are endocrinally modulated, and that their alteration by candidate endocrine disruptors, such as MTC and DDT, constitutes a possible pathway of the harmful effects of these components on fetal development.

The ZF87/MAZ transcription factor functions as a growth suppressor in fibroblasts.

ZF87/MAZ is a zinc finger transcription factor that activates expression of tissue-specific genes and represses expression of the c-myc proto-oncogene. Infection of NIH3T3 fibroblasts with a retrovirus expressing ZF87/MAZ leads to a significant reduction in G418-resistant colonies, compared to cells infected with a retroviral control. Further, only a small fraction of the G418-resistant colonies express ZF87/MAZ. When the ZF87/MAZ-expressing colonies are expanded, they demonstrate a slow growth phenotype, a delayed transit through G1 phase and a decrease in endogenous c-myc gene expression and cyclin A and cyclin E protein levels. Consistent with a partial G1 arrest, the ZF87/MAZ-expressing cells show a reduced sensitivity to the S phase specific chemotherapeutic agent camptothecin. These data indicate that ZF87/MAZ is a growth suppressor protein in nontransformed cells, in part, by affecting the levels of key cell cycle regulatory proteins.

The N-terminal domain of the mineralocorticoid receptor modulates both mineralocorticoid receptor- and glucocorticoid receptor-mediated transactivation from Na/K ATPase beta1 target gene promoter.

Mineralocorticoid and glucocorticoid hormones activate the expression of the Na/K ATPase beta1 through direct binding of the mineralocorticoid receptor (MR) and glucocorticoid receptors (GR) to a mineralocorticoid- and glucocorticoid-responsive element in the beta1 promoter region, but activation of the beta1 promoter is inhibited by coexpression of both receptors. Here, using a series of mutated and chimeric receptors, we show that the N-terminal region of MR mediates an inhibitory effect on MR and GR activation from the beta1 promoter, in CV-1 cells. Deletion of the N-terminal region of MR (1-603) enhanced MR activation four-fold. Activation by chimeric MR, in which the N-terminus of GR replaces the N-terminal region of MR, was threefold that of wild-type MR. In addition, whereas coexpression of wild-type MR and GR was inhibitory, coexpression of chimeric MR and wild-type GR was nearly equal to that of MR. By contrast, mutated GR lacking its N-terminal region (1-420) was less efficient than the wild type in activating this promoter. These results demonstrate that the N-terminal domains of MR and GR have opposite transactivation properties and that MR region 1-603 is indeed inhibitory for both MR- and GR-mediated regulation of the Na/K ATPase beta1 gene promoter.

Regulation of the human Na/K-ATPase beta1 gene promoter by mineralocorticoid and glucocorticoid receptors.

Expression of the human Na/K-ATPase beta1 subunit is regulated by a mineralocorticoid- and glucocorticoid-responsive elements. Here we identified an MR and GR responsive element, at positions -650 to -630, within the beta1 gene promoter that is required for both MR and GR activation. Independent expression of MR and GR activated by aldosterone or triamcinolone acetonide (TA) leads to significant transactivation of the beta1 promoter. Yet coexpression of both receptors activated by aldosterone plus TA or cortisol results in a much lower induction, indicating that coexpression of MR and GR is inhibitory. Gel shift mobility assay using an oligonucleotide including the 21-base pair MRE/GRE with whole cell extracts prepared from CV-1 cells overexpressing MR or GR showed specific MR and GR binding to this sequence. Additionally, antibodies to both MR and GR effectively supershifted the protein-DNA complexes, indicating that these receptors bound to the DNA sequence. Finally, the 21-base pair MRE/GRE was capable of activating transcription from a heterologous promoter in response to both aldosterone and TA. Together these data indicate that the 21-base pair sequence represents a true MRE/GRE and that optimal activation of the human Na/K-ATPase beta1 promoter is controlled by mineralocorticoid and glucocorticoid hormones. It appears that an interaction of MR with GR on the beta1 promoter effectively down-regulates transcription.

Reduction in fibronectin expression and alteration in cell morphology are coincident in NIH3T3 cells expressing a mutant E2F1 transcription factor.

Fibronectin within the extracellular matrix plays a role in cell attachment, spreading, and shape, while it also affects aspects of cell proliferation. Transcription factors such as E2F1 are also known to regulate cell shape and cell proliferation. Yet, to date no linkage has been established between fibronectin expression and E2F1. We show here that cells constitutively expressing a mutant E2F1 protein (E2F1d87) produce reduced amounts of fibronectin mRNA and protein. The altered expression of fibronectin seen in the E2F1d87 expressing cells is due, in part, to a reduction in transcription from the fibronectin promoter. Providing exogenous fibronectin, but not Type I collagen or laminin, as a substrate for cell adhesion is sufficient to revert the altered morphology and reestablish actin-containing microfilaments lost in the mutant cell line. An additional characteristic of the cells expressing the mutant E2F1 is that they demonstrate slow growth and a doubling in S phase duration. While providing exogenous fibronectin as an adhesion substrate did not shorten the S phase duration in the mutant line, it did significantly shorten the S phase duration in the parental NIH3T3 cell line, implicating a role for the extracellular matrix in regulating S phase transit in normal cells.

The unliganded mineralocorticoid receptor is associated with heat shock proteins 70 and 90 and the immunophilin FKBP-52.

The human mineralocorticoid receptor (MR) is a member of the steroid-thyroid hormone receptor superfamily, which includes receptors for retinoic acid, vitamin D, and other steroids, such as the glucocorticoids (which bind the glucocorticoid receptor, GR). MR and GR, the corticosteroid receptors, share significant homology and are activated by steroid binding, resulting in a conformational change, nuclear translocation, and DNA binding. Despite these similarities with GR, the MR remains less well characterized. However, protein components known to be present in the unliganded GR are also likely to be components of the heteromeric MR complex. In the current study, we investigated whether or not hsp70, hsp90, and the immunophilin FKBP-52 are present in the nonsteroid-bound MR complex, because these proteins are known to be present in the unliganded GR complex. The unliganded MR complex was assembled in vitro using reticulocyte lysate and in vivo using the baculovirus overexpression system and Spodoptera frugiperda (Sf9) cells. Western blot analysis revealed the presence of hsp70, hsp90, and FKBP-52 in the unliganded complexes, but hsp90 and FKBP-52 were not detected following exposure to aldosterone. Electrophoretic mobility shift analysis demonstrated that DNA binding of MR occurred only after treatment with aldosterone. These studies indicate that proteins associated with the unliganded GR are also present in the unliganded MR complex, and that hsp90 and FKBP-52 dissociate prior to DNA binding in a manner similar to that described for GR. Finally, the stoichiometric analysis of the proteins present within the heteromeric MR complex suggests a divergence between this receptor and the GR.