Superficial and deep zone articular chondrocytes exhibit differences in actin polymerization status and actin-associated molecules in vitro.

Objective: The actin cytoskeleton regulates cell shape and plays a role in regulating chondrocyte phenotype. Most studies investigating regulation of the chondrocyte phenotype by the actin cytoskeleton use chondrocytes isolated from full-thickness (FT) cartilage, which has a heterogeneous cell population. Superficial zone chondrocytes (SZC) have an elongated morphology and account for 10-20% of chondrocytes, while the remaining chondrocytes in the deeper zones appear more rounded. This study characterizes the actin cytoskeleton and expression of actin-associated molecules in SZC and deep zone (DZ) chondrocytes (DZC) in vitro in order to identify molecules differentially expressed by SZC and DZC that may contribute to the observed differences in zonal chondrocyte shapes. Design: SZ, DZ, and FT chondrocytes isolated from bovine metacarpal-phalangeal joints were cultured in monolayer for 48 h. Macroscopic morphology, actin polymerization status, and expression of select actin-associated molecules (adseverin, cofilin, transgelin, vinculin, MRTF-A, and YAP/TAZ) were determined. Results: SZC appeared more elongated and have more filamentous actin compared to DZC, as determined by quantifying cell circularity and G-/F-actin ratio. MRTF-A gene and protein levels were significantly higher in SZC compared to DZC while DZC more highly expressed transgelin and TAZ. Although there was differential gene expression, no significant differences in adseverin, cofilin, vinculin, or YAP protein levels were observed between the two cell populations. Conclusions: This study identifies differences in actin polymerization status and expression of actin-associated molecules in primary SZC and DZC in vitro. These findings further our understanding of candidate actin-related pathways that may be regulating zonal chondrocyte phenotype.

CDC42 regulates the expression of superficial zone molecules in part through the actin cytoskeleton and myocardin-related transcription factor-A.

Osteoarthritis (OA) is a degenerative disease that initially manifests as loss of the superficial zone (SZ) of articular cartilage. SZ chondrocytes (SZC) differ in morphology from other chondrocytes as they are elongated and oriented parallel to the tissue surface. Proteoglycan 4 (PRG4) and tenascin C (TNC) are molecules expressed by SZC, which have been shown to be chondroprotective. Identification of the signalling pathway(s) regulating expression of SZ molecules may lead to a therapeutic target that can be used to delay or prevent the onset of OA. The hypothesis of this study is that expression of SZ molecules are regulated in part, by the CDC42-actin-myocardin-related transcription factor-A (MRTF-A) signaling pathway. SZC from bovine metacarpal-phalangeal joints were isolated and grown in monolayer culture. Each target in the CDC42-actin-MRTF-A pathway was inhibited and the effect on cell shape, actin cytoskeleton status, and expression of PRG4 and TNC were determined. Treatment with the CDC42 inhibitor ML141 decreased PRG4 and TNC expression, and correlated with increased cell circularity and G-/F-actin ratio. PRG4 and TNC expression were differentially regulated by actin depolymerizing agents, latrunculin B and cytochalasin D. Chemical inhibition of MRTF-A resulted in decreased expression of both PRG4 and TNC; however, specific knockdown by small interfering RNA only decreased expression of TNC indicating that TNC, but not PRG4, is regulated by MRTF-A. Although PRG4 and TNC expression are both regulated by CDC42 and actin, it appears to occur through different downstream signaling pathways. Further study is required to elucidate the pathway regulating PRG4. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2421-2430, 2018.

Efficient, Low-Cost Nucleofection of Passaged Chondrocytes.

Nucleofection of chondrocytes has been shown to be an adequate method of transfection. Using Amaxa's nucleofection system, transfection efficiencies up to 89% were achievable for vector (pmaxGFP) and 98% for siRNA (siGLO) into passaged chondrocytes. However, such methods rely on costly commercial kits with proprietary reagents limiting its use in basic science labs and in clinical translation. Bovine-passaged chondrocytes were plated in serum reduced media conditionsand then nucleofected using various in laboratory-produced buffers. Cell attachment, confluency, viability, and transfection efficiency was assessed following nucleofection. For each parameter the buffers were scored and a final rank for each buffer was determined. Buffer denoted as 1M resulted in no significant difference for cell attachment, confluency, and viability as compared to non-nucleofected controls. Nucleofection in 1M buffer, in the absence of DNA vectors, resulted in increased col2, ki67, ccnd1 mRNA levels, and decreased col1 mRNA levels at 4 days of culture. Flow cytometry revealed that the transfection efficiency of 1M buffer was comparable to that obtained using the Amaxa commercial kit. siRNA designed against lamin A/C resulted in an average reduction of lamin A and C proteins to 19% and 8% of control levels, respectively. This study identifies a cost-effective, efficient method of nonviral nucleofection of bovine-passaged chondrocytes using known buffer formulations. Human-passaged chondrocytes could also be successfully nucleofected in 1M buffer. Thus this method should facilitate cost-efficient gene targeting of cells used for articular cartilage repair in a research setting.

Expression of type I collagen and tenascin C is regulated by actin polymerization through MRTF in dedifferentiated chondrocytes.

This study examined actin regulation of fibroblast matrix genes in dedifferentiated chondrocytes. We demonstrated that dedifferentiated chondrocytes exhibit increased actin polymerization, nuclear localization of myocardin related transcription factor (MRTF), increased type I collagen (col1) and tenascin C (Tnc) gene expression, and decreased Sox9 gene expression. Induction of actin depolymerization by latrunculin treatment or cell rounding, reduced MRTF nuclear localization, repressed col1 and Tnc expression, and increased Sox9 gene expression in dedifferentiated chondrocytes. Treatment of passaged chondrocytes with MRTF inhibitor repressed col1 and Tnc expression, but did not affect Sox9 expression. Our results show that actin polymerization regulates fibroblast matrix gene expression through MRTF in passaged chondrocytes.