Dedifferentiation and redifferentiation of articular chondrocytes from surface and middle zones: changes in microRNAs-221/-222, -140, and -143/145 expression.

Articular cartilage contains three functional zones (superficial, middle, and deep) characterized by distinct structure, composition, and biomechanical properties. One of the unsolved major challenges in cartilage tissue engineering is to produce tissue that mimics the zonal organization of the native articular cartilage. An increasing number of studies aim to design zonal organization into tissue-engineered cartilage by forming a stratified construct using zonal cell subpopulations. However, in vitro monolayer expansion of chondrocytes, which is generally required to obtain high cell numbers necessary for tissue engineering and autologous chondrocyte implantation, leads to dedifferentiation of chondrocytes into fibroblast-like cells, resulting in loss of zonal markers, such as the superficial zone protein (SZP) of the superficial zone as well as chondrocytic phenotype markers, such as type II collagen and aggrecan. Several microRNAs (miRNAs), including miR-221, miR-222, miR-143, and miR-145, have been identified from bovine articular cartilage as superficial zone-enriched miRNAs. miR-140 has been known as a cartilage-specific miRNA whose expression is implicated in chondrocyte differentiation and cartilage tissue homeostasis. As miRNAs play an important role in regulating gene expression during cell differentiation and maintaining tissue homeostasis, we determined the expression of the miRNAs with zonal differentiation and homeostasis. We investigated how chondrocyte dedifferentiation during multiple passages and redifferentiation in a three-dimensional (3D) agarose culture regulates the expression of these miRNAs by quantitative reverse transcription-polymerase chain reaction. Additionally, the effect of transforming growth factor beta 1 (TGF-ß1), which is known to enhance chondrocytic differentiation and SZP expression, on these miRNAs was evaluated. The expression of miR-221 and miR-222 increased during dedifferentiation and during redifferentiation in a 3D culture and TGF-ß1 restored them to normalcy. miR-140 dramatically decreased during dedifferentiation and its expression is partially recovered in a 3D culture with TGF-ß1. miR-143 and miR-145 in the superficial chondrocytes decreased during dedifferentiation and further decreased in a 3D culture, but TGF-ß1 partially recovered their expression in a 3D culture. In conclusion, the expression patterns of the miRNAs will be of functional utility for strategies and approaches to tissue engineering of the articular cartilage.

c-Maf Transcription Factor Regulates ADAMTS-12 Expression in Human Chondrogenic Cells.

OBJECTIVE: ADAMTS (a disintegrin and metalloproteinase with thrombospondin type-1 motif) zinc metalloproteinases are important during the synthesis and breakdown of cartilage extracellular matrix. ADAMTS-12 is up-regulated during in vitro chondrogenesis and embryonic limb development; however, the regulation of ADAMTS-12 expression in cartilage remains unknown. The transcription factor c-Maf is a member of Maf family of basic ZIP (bZIP) transcription factors. Expression of c-Maf is highest in hypertrophic chondrocytes during embryonic development and postnatal growth. We hypothesize that c-Maf and ADAMTS-12 are co-expressed during chondrocyte differentiation and that c-Maf regulates ADAMTS-12 expression during chondrogenesis. DESIGN: Promoter analysis and species alignments identified potential c-Maf binding sites in the ADAMTS-12 promoter. c-Maf and ADAMTS-12 co-expression was monitored during chondrogenesis of stem cell pellet cultures. Luciferase expression driven by ADAMTS-12 promoter segments was measured in the presence and absence of c-Maf, and synthetic oligonucleotides were used to confirm specific binding of c-Maf to ADAMTS-12 promoter sequences. RESULTS: In vitro chondrogenesis from human mesenchymal stem cells revealed co-expression of ADAMTS-12 and c-Maf during differentiation. Truncation and point mutations of the ADAMTS-12 promoter evaluated in reporter assays localized the response to the proximal 315 bp of the ADAMTS-12 promoter, which contained a predicted c-Maf recognition element (MARE) at position -61. Electorphoretic mobility shift assay confirmed that c-Maf directly interacted with the MARE at position -61. CONCLUSIONS: These data suggest that c-Maf is involved in chondrocyte differentiation and hypertrophy, at least in part, through the regulation of ADAMTS-12 expression at a newly identified MARE in its proximal promoter.

MicroRNAs in chondrogenesis, articular cartilage, and osteoarthritis: implications for tissue engineering.

Coordinated actions of various regulators, including morphogens are required for chondrogenesis and maintenance of articular cartilage function. Bone morphogenetic proteins, and related signaling molecules and transcription factors form a complex regulatory network. MicroRNAs (miRNAs) are noncoding small RNAs that negatively regulate the expression of downstream targets by repressing the translation or inducing the cleavage of messenger RNAs (mRNAs). Increasing evidence indicates that miRNAs are an integral part of the regulatory network in chondrocyte differentiation and cartilage function. The aim of this article is to review the progress in miRNA expression and target genes in cartilage differentiation, homeostasis, and in the pathobiology of osteoarthritis. The recent progress in miRNAs in cartilage has implications for tissue engineering.

Enhanced activity of transforming growth factor ß1 (TGF-ß1) bound to cartilage oligomeric matrix protein.

Cartilage oligomeric matrix protein (COMP) is an important non-collagenous cartilage protein that is essential for the structural integrity of the cartilage extracellular matrix. The repeated modular structure of COMP allows it to "bridge" and assemble multiple cartilage extracellular matrix components such as collagens, matrilins, and proteoglycans. With its modular structure, COMP also has the potential to act as a scaffold for growth factors, thereby affecting how and when the growth factors are presented to cell-surface receptors. However, it is not known whether COMP binds growth factors. We studied the binding interaction between COMP and TGF-ß1 in vitro and determined the effect of COMP on TGF-ß1-induced signal transduction in reporter cell lines and primary cells. Our results demonstrate that mature COMP protein binds to multiple TGF-ß1 molecules and that the peak binding occurs at slightly acidic pH. These interactions were confirmed by dual polarization interferometry and visualized by rotary shadow electron microscopy. There is cation-independent binding of TGF-ß1 to the C-terminal domain of COMP. In the presence of manganese, an additional TGF-ß-binding site is present in the TSP3 repeats of COMP. Finally, we show that COMP-bound TGF-ß1 causes increased TGF-ß1-dependent transcription. We conclude that TGF-ß1 binds to COMP and that TGF-ß1 bound to COMP has enhanced bioactivity.

Role of c-Maf in Chondrocyte Differentiation: A Review.

Chondrocyte differentiation in the growth plate is an important process for the longitudinal growth of endochondral bones. Sox9 and Runx2 are the most often-studied transcriptional regulators of the chondrocyte differentiation process, but the importance of additional factors is also becoming apparent. Mafs are a subfamily of the basic ZIP (bZIP) transcription factor superfamily, which act as key regulators of tissue-specific gene expression and terminal differentiation in many tissues. There is increasing evidence that c-Maf and its splicing variant Lc-Maf play a role in chondrocyte differentiation in a temporal-spatial manner. This review summarizes the functions of c-Maf in chondrocyte differentiation and discusses the possible role of c-Maf in osteoarthritis progression.

A La-related protein modulates 7SK snRNP integrity to suppress P-TEFb-dependent transcriptional elongation and tumorigenesis.

The general transcription factor P-TEFb stimulates RNA polymerase II elongation and cotranscriptional processing of pre-mRNA. Contributing to a functional equilibrium important for growth control, a reservoir of P-TEFb is maintained in an inactive snRNP where 7SK snRNA is a central scaffold. Here, we identify PIP7S as a La-related protein stably associated with and required for 7SK snRNP integrity. PIP7S binds and stabilizes nearly all the nuclear 7SK via 3' -UUU-OH, leading to the sequestration and inactivation of P-TEFb. This function requires its La domain and intact C terminus. The latter is frequently deleted in human tumors due to microsatellite instability-associated mutations. Consistent with the tumor suppressor role of a Drosophila homolog of PIP7S, loss of PIP7S function shifts the P-TEFb equilibrium toward the active state, disrupts epithelial differentiation, and causes P-TEFb-dependent malignant transformation. Through PIP7S modulation of P-TEFb, our data thus link a general elongation factor to growth control and tumorigenesis.

Alphavirus capsid protein helix I controls a checkpoint in nucleocapsid core assembly.

The assembly of the alphavirus nucleocapsid core has been investigated using an in vitro assembly system. The C-terminal two-thirds of capsid protein (CP), residues 81 to 264 in Sindbis virus (SINV), have been previously shown to have all the RNA-CP and CP-CP contacts required for core assembly in vitro. Helix I, which is located in the N-terminal dispensable region of the CP, has been proposed to stabilize the core by forming a coiled coil in the CP dimer formed by the interaction of residues 81 to 264. We examined the ability of heterologous alphavirus CPs to dimerize and form phenotypically mixed core-like particles (CLPs) using an in vitro assembly system. The CPs of SINV and Ross River virus (RRV) do not form phenotypically mixed CLPs, but SINV and Western equine encephalitis virus CPs do form mixed cores. In addition, CP dimers do not form between SINV and RRV in these assembly reactions. In contrast, an N-terminal truncated SINV CP (residues 81 to 264) forms phenotypically mixed CLPs when it is assembled with full-length heterologous CPs, suggesting that the region that controls the mixing is present in the N-terminal 80 residues. Furthermore, this result suggests that the dimeric interaction, which was absent between SINV and RRV CPs, can be restored by the removal of the N-terminal 80 residues of the SINV CP. We mapped the determinant that is responsible for phenotypic mixing onto helix I by using domain swapping experiments. Thus, discrimination of the CP partner in alphavirus core assembly appears to be dependent on helix I sequence compatibility. These results suggest that helix I provides one of the important interactions during nucleocapsid core formation and may play a regulatory role during the early steps of the assembly process.

In vitro-assembled alphavirus core-like particles maintain a structure similar to that of nucleocapsid cores in mature virus.

In vitro-assembled core-like particles produced from alphavirus capsid protein and nucleic acid were studied by cryoelectron microscopy. These particles were found to have a diameter of 420 A with 240 copies of the capsid protein arranged in a T=4 icosahedral surface lattice, similar to the nucleocapsid core in mature virions. However, when the particles were subjected to gentle purification procedures, they were damaged, preventing generation of reliable structural information. Similarly, purified nucleocapsid cores isolated from virus-infected cells or from mature virus particles were also of poor quality. This suggested that in the absence of membrane and glycoproteins, nucleocapsid core particles are fragile, lacking accurate icosahedral symmetry.