The ubiquitin-proteasome pathway regulates the turnover of many transcription factors, including steroid hormone receptors such as the estrogen receptor and progesterone receptor. For these receptors, proteasome inhibition interferes with steroid-mediated transcription. We show here that proteasome inhibition with MG132 results in increased accumulation of the glucocorticoid receptor (GR), confirming that it is likewise a substrate for the ubiquitin-proteasome degradative pathway. Using the mouse mammary tumor virus (MMTV) promoter integrated into tissue culture cells, we found that proteasome inhibition synergistically increases GR-mediated transactivation. This increased activation was observed in a number of cell lines and on various MMTV templates, either as transiently transfected reporters or stably integrated into chromatin. These observations suggest that the increase in GR-mediated transcription due to proteasome inhibition may occur downstream of the initial chromatin remodeling step. In support of this concept, the increase in transcription did not correlate with an increase in chromatin remodeling, as measured by restriction enzyme hypersensitivity, or transcription factor loading, as exemplified by nuclear factor 1. To investigate the relationship between GR turnover, transcription, and subnuclear trafficking, we examined the effect of proteasome inhibition on the mobility of the GR within the nucleus and association of the GR with the nuclear matrix. Blocking GR turnover reduced the mobility of the GR within the nucleus, and this correlated with increased association of the receptor with the nuclear matrix. As a result of proteasome inhibition, GR mobility within the nucleus was reduced while its association with the nuclear matrix was increased. Thus, while altered nuclear mobility of steroid receptors may be a common feature of proteasome inhibition, GR is unique in its enhanced transactivation activity that results when proteasome function is compromised. Proteasomes may therefore impact steroid receptor action at multiple levels and exert distinct effects on individual receptor types.Glucocorticoids mediate diverse cellular processes such as differentiation, development, and homeostasis through the ligand-activated glucocorticoid receptor (GR) (24). The GR is a versatile transcriptional regulator, and it can activate or repress a large variety of natural promoters in multiple tissues. This versatility is due in part to the numerous mechanisms utilized by the GR to regulate transcription, depending on the cellular and promoter context. These mechanisms include GR modification of chromatin structure through interactions with chromatin remodeling complexes, differential binding of the receptor to both canonical and noncanonical response elements, binding to other regulatory factors such as AP-1 and NF-B, and interaction with coactivators (2,11,46).The activity of the GR is also subject to regulation that occurs at least in part by changes in the half-life of the receptor protein (6). Turnover of t...

The aim of this study was to evaluate an embroidered polycaprolactone-co-lactide (trade name PCL) scaffold for the application in bone tissue engineering. The surface of the PCL scaffolds was hydrolyzed with NaOH and coated with collagen I (coll I) and chondroitin sulfate (CS). It was investigated if a change of the surface properties and the application of coll I and CS could promote cell adhesion, proliferation, and osteogenic differentiation of human mesenchymal stem cells (hMSC). The porosity (80%) and pore size (0.2-1 mm) of the scaffold could be controlled by embroidery technique and should be suitable for bone ingrowth. The treatment with NaOH made the polymer surface more hydrophilic (water contact angle dropped to 25%), enhanced the coll I adsorption (up to 15%) and the cell attachment (two times). The coll I coated scaffold improved cell attachment and proliferation (three times). CS, as part of the artificial matrix, could induce the osteogenic differentiation of hMSC without other differentiation additives. The investigated scaffolds could act not just as temporary matrix for cell migration, proliferation, and differentiation in bone tissue engineering but also have a great potential as bioartificial bone substitute.

To investigate and assess bone regeneration in sheep in combination with new implant materials classical histological staining methods as well as immunohistochemistry may provide additional information to standard radiographs or computer tomography. Available published data of bone defect regenerations in sheep often present none or sparely labeled histological images. Repeatedly, the exact location of the sample remains unclear, detail enlargements are missing and the labeling of different tissues or cells is absent. The aim of this article is to present an overview of sample preparation, staining methods and their benefits as well as a detailed histological description of bone regeneration in the sheep tibia. General histological staining methods like hematoxylin and eosin, Masson-Goldner trichrome, Movat's pentachrome and alcian blue were used to define new bone formation within a sheep tibia critical size defect containing a polycaprolactone-co-lactide (PCL) scaffold implanted for 3 months (n = 4). Special attention was drawn to describe the bone healing patterns down to cell level. Additionally one histological quantification method and immunohistochemical staining methods are described.

The current study was undertaken with the goal being isolation, cultivation, and characterization of ovine mesenchymal stem cells (oMSC). Furthermore, the objective was to determine whether biological active polycaprolactone-co-lactide (trade name PCL) scaffolds support the growth and differentiation of oMSC in vitro. The oMSC were isolated from the iliac crest of six merino sheep. Three factors were used to demonstrate the MSC properties of the isolated cells in detail. (1) Their ability to proliferate in culture with a spindle-shaped morphology, (2) presence of specific surface marker proteins, and (3) their capacity to differentiate into the three classical mesenchymal pathways, osteoblastic, adipogenic, and chondrogenic lineages. Furthermore, embroidered PCL scaffolds were coated with collagen I (coll I) and chondroitin sulfate (CS). The porous structure of the scaffolds and the coating with coll I/CS allowed the oMSC to adhere, proliferate, and to migrate into the scaffolds. The coll I/CS coating on the PCL scaffolds induced osteogenic differentiation of hMSC, without differentiation supplements, indicating that the scaffold also has an osteoinductive character. In conclusion, the isolated cells from the ovine bone marrow have similar morphologic, immunophenotypic, and functional characteristics as their human counterparts. These cells were also found to differentiate into multiple mesenchymal cell types. This study demonstrates that embroidered PCL scaffolds can act as a temporary matrix for cell migration, proliferation, and differentiation of oMSC. The data presented will provide a reliable model system to assess the translation of MSC-based therapy into a variety of valuable ovine experimental models under autologous settings.