Effects of mechanical strain on ANK, ENPP1 and TGF-β1 expression in rat endplate chondrocytes in�vitro

Abstract

Abstract. We investigated the effects of mechanical strain on the progressive ankylosis (ANK) gene and extracellular nucleotide phosphatase/phosphodiesterase (ENPP)1 mRNA expression and TGF-β1 protein expression in rat endplate chondrocytes in vitro. Endplate chondrocytes were isolated and cultured in vitro. Following identification with toluidine blue and immunocytochemical staining, chondrocytes were subjected to 10% elongation with various frequencies (0.5, 1, 1.5 and 2 Hz) using a Flexercell Tension Plus system at various intervals (3, 6, 12, 24, 36 and 48 h). As a control, cells that had been cultured statically on the same type of plate but were not subjected to stretch were also observed. Real-time reverse transcription-polymerase chain reaction and the enzyme-linked immunosorbent assay were used to study the effects of mechanical strain on ANK and ENPP1 mRNA expression and TGF-β1 concentration in the supernatant, respectively. Following treatment, the shape of the chondrocytes displayed a significant change from the original polygon to a typical spindle cell morphology; and the arrangement of the cells exhibited a change from a haphazard arrangement to an alignment with a certain direction. In the 0.5 Hz, 24-h group, the ANK gene expression was significantly increased compared to the control group (P<0.05); whereas in the other groups, the ANK and ENPP1 expression levels were reduced. With the increased frequencies in the 24-h group, the ANK gene expression gradually reduced. Changes in the expression of ANK and ENPP1 followed similar trends. TGF-β1 in the supernatant increased gradually in each frequency group, with a clear increase in the 0.5 Hz group. We conclude that various frequencies of mechanical strain can affect the expression of ANK, ENPP1 and endogenous TGF-β1 in endplate chondrocytes. Our results indicate that 0.5 Hz, 24 h may be the optimal stimulation condition to prevent calcification occurrence and to maintain the function of endplate chondrocytes.

Degeneration and calcification of the cervical endplate is connected with decreased expression of ANK, ENPP-1, OPN and TGF-β1 in the intervertebral disc

“…It has been mentioned [9] that when the ANK channel protein fails in its function, then calcium pyrophosphate dehydrate is deposited in the cartilage. The current study confirms earlier findings from animal models that ANK protein expression decreases with degeneration [10], suggesting its significant role in the mechanism of pathological calcification. It has been reported that TGF-b1 plays an important role in regulating the expression of ANK in chondrocytes, through Ras/Raf-1/ERK and Ca…”

“…It produces a multiple-pass transmembrane protein (ANK) which regulates intracellular inorganic pyrophosphate (PPi) transportation from the cytoplasm to the extracellular space, thereby maintaining the PPi steady-state concentration, and thus possibly preventing increased calcification of the tissue [10]. Findings from previous studies show that mRNA and protein products of ANKH decrease with endplate degeneration [11].…”

Abnormal Mechanical Loading Induces Cartilage Degeneration by Accelerating Meniscus Hypertrophy and Mineralization After ACL Injuries In Vivo

“…ANKH regulates extracellular PPi steady-state concentrations by transporting PPi to the extracellular microenvironment. 46 ENPP1 increases the extracellular PPi concentration to effectively inhibit HA crystals and plays a significant role in regulating physiological and pathological tissue mineralization. 33 ALP increases Pi concentrations by cleaving PPi.…”

Intermittent Cyclic Mechanical Tension-Induced Calcification and Downregulation of ankh Gene Expression of End Plate Chondrocytes

“…The specifi c method is reported in our last article. 7 The second passage cells were used for experiments.…”