Cytoskeleton Aberrations in Alkaptonuric Chondrocytes

Abstract

Alkaptonuria (AKU) is an ultra-rare autosomal genetic disorder caused by a defect in the activity of the enzyme homogentisate 1,2-dioxygenase (HGD) that leads to the accumulation of homogentisic acid (HGA) and its oxidized product, benzoquinone acetic acid (BQA), in the connective tissues causing a pigmentation called "ochronosis." The consequent progressive formation of ochronotic aggregates generate a severe condition of oxidative stress and inflammation in all the affected areas. Experimental evidences have also proved the presence of serum amyloid A (SAA) in several AKU tissues and it allowed classifying AKU as a secondary amyloidosis. Although AKU is a multisystemic disease, the most affected system is the osteoarticular one and articular cartilage is the most damaged tissue. In this work, we have analyzed for the first time the cytoskeleton of AKU chondrocytes by means of immunofluorescence staining. We have shown the presence of SAA within AKU chondrocytes and finally we have demonstrated the co-localization of SAA with three cytoskeletal proteins: actin, vimentin, and β-tubulin. Furthermore, in order to observe the ultrastructural features of AKU chondrocytes we have performed TEM analysis, focusing on the Golgi apparatus structure and, to demonstrate that pigmented areas in AKU cartilage are correspondent to areas of oxidation, 4-HNE presence has been evaluated by means of immunofluorescence. J. Cell. Physiol. 232: 1728-1738, 2017. © 2016 Wiley Periodicals, Inc.

Reduced primary cilia length and altered Arl13b expression are associated with deregulated chondrocyte Hedgehog signaling in alkaptonuria

“…In the present study, we report variations in primary cilia prevalence and length for AKU chondrocytes which may be associated with alterations in the organization of actin (Fig. ) and other cytoskeletal components (Geminiani et al, ). The filamentous microtubule structure is transformed in AKU chondrocytes, forming aggregates at the cell periphery (Geminiani et al, ).…”

“…Similarly, Geminiani et al (2016) recently reported that both actin and microtubule cytoskeletal elements are disordered in AKU chondrocytes. In addition to alterations in cell shape, actin stress fibre formation is increased in AKU chondrocytes accompanied by the formation of actin bundles in the nuclear periphery (Geminiani et al, 2016). The disruption of actin organization and dynamics may have consequences for the maintenance of chondrocyte phenotype (Brown & Benya, 1988), mechanotransduction (Ohashi, Hagiwara, Bader, & Knight, 2006;Wright et al, 1997), and may also influence chondrocyte function through the primary cilium.…”

“…1) and other cytoskeletal components (Geminiani et al, 2016). The filamentous microtubule structure is transformed in AKU chondrocytes, forming aggregates at the cell periphery (Geminiani et al, 2016). Furthermore, the vimentin network appears granular in these cells resulting in alterations in cell shape and cell shrinkage.…”

“…It is well established that the cytoskeleton is disrupted in OA chondrocytes (Capin-Gutierrez, Talamas-Rohana, Gonzalez-Robles, Lavalle- Montalvo, & Kouri, 2004;Holloway et al, 2004;Haudenschild et al, 2011). Similarly, Geminiani et al (2016) recently reported that both actin and microtubule cytoskeletal elements are disordered in AKU chondrocytes. In addition to alterations in cell shape, actin stress fibre formation is increased in AKU chondrocytes accompanied by the formation of actin bundles in the nuclear periphery (Geminiani et al, 2016).…”

Smoothened‐antagonists reverse homogentisic acid‐induced alterations of Hedgehog signaling and primary cilium length in alkaptonuria

“…There are numerous findings indicating that, among the processes that lead to cartilage degradation in AKU, important roles are played by oxidative stress (Braconi et al, , , ), inflammation (Millucci et al, ), and accumulation of serum amyloid A (SAA), describing AKU as a secondary amyloidosis (Millucci et al, ,, ). More recently, the structural analysis of AKU chondrocyte cytoskeleton revealed that the organization of the three main cytoskeletal proteins (tubulin, actin, and vimentin) is altered in AKU cells (Geminiani et al, ) and that chondrocytes cultured in HGA‐added medium started developing the same alterations observed in AKU chondrocytes. Alterations in cytoskeletal tension have previously been shown to regulate the structure and function of primary cilia (McMurray, Wann, Thompson, Connelly, & Knight, ; Pitaval, Tseng, Bornens, & Thery, ).…”