Table 1.
Primers used for quantitative RT-PCR.
Fig 1.
EGR1 overexpression prevents the downregulation of tendon-associated gene expression in tension released engineered tendons.
(A) Two week-old fibrin gel constructs made of mouse C3H10T1/2 cells or C3H10T1/2-EGR1 cells were analyzed for tendon gene expression by RT-q-PCR analyses. The mRNA levels of C3H10T1/2 constructs were normalized to 1. Errors bars represent standard errors of the mean of 5 C3HT101/2 constructs and 7 C3H10T1/2-EGR1 constructs. The p values were calculated using the Mann-Whitney test. The mRNA levels of Egr1, Scx, Tnmd and Tgfb2 genes were increased in C3H10T1/2-EGR1 constructs compared to those of C3H10T1/2 constructs. (B) Tension was released in C3H10T1/2 constructs by sectioning one end of the construct. Transcript levels were analyzed by RT-q-PCR analyses in tension-released C3H10T1/2 constructs and compared to C3H10T1/2 constructs. The mRNA levels of C3H10T1/2 constructs were normalized to 1. Errors bars represent standard errors of the mean of 7 C3H10T1/2 constructs and 7 tension-released C3H10T1/2 constructs. The p values were calculated using the Mann-Whitney test. We observed a decrease in the transcript levels of Egr1, Scx, Tnmd, Col1a1, Col1a2 and Tgfb2 genes in tension-released C3H10T1/2 constructs (TR) compared to tensioned C3H10T1/2 constructs (T). (C) The mRNA levels of tendon genes were analyzed in tension-released C3H10T1/2-EGR1 constructs and compared with tensioned C3H10T1/2-EGR1 constructs by RT-q-PCR analyses. The mRNA levels of C3H10T1/2-EGR1 constructs were normalized to 1. Errors bars represent standard errors of the mean of 7 C3HT101/2-EGR1 constructs and 5 tension-released C3H10T1/2-EGR1 constructs. The p values were calculated using the Mann-Whitney test. There was no significant change in tendon gene expression in tension-released C3H10T1/2-EGR1 constructs (TR) compared to tensioned C3H10T1/2-EGR1 constructs (T). T, Tension, TR, Tension release.
Fig 2.
Reduced mechanical input induces a diminution of Egr1 and Scx expression in adult tendons.
(A) Botox or physiological saline injections in Gastrocnemius muscles of adult mice. (B,C) LacZ staining (reflecting Egr1 expression) in tendons, 1 week following physiological saline or Botox injection in Egr1Lacz/+ adult mice. (D) RT-q-PCR analyses of tendons, one or two weeks after Botox or physiological saline injections in muscles in adult mice. The mRNA levels of tendons following Botox injection were compared to those of tendons with physiological saline injection. Errors bars represent the standard deviations of 5 (one week) and 7 (two weeks) biological samples. The mRNA levels of Egr1, Scx, Col1a2, Tgfb2 genes were decreased one or two weeks after Botox injection compared to physiological saline injection. Tnmd mRNA levels were not significantly decreased after Botox injection. The p values were calculated using the Wilcoxon test.
Fig 3.
Mechanical signals are required for normal tendon gene response following tendon injury.
(A) Botox or physiological saline injections in Gastrocnemius muscles and tendon injury in adult mice. (B) RT-q-PCR analyses of tendons, 2 weeks after tendon injury and Botox injection in muscles. The mRNA levels of tendons following injury and physiological saline injection in muscles were normalized to 1. The errors bars represent the standard error of the means of 10 and 11 biological samples of injured tendons after physiological saline or Botox injections, respectively. The p values were calculated using the Mann-Whitney test. The mRNA levels of the Egr1, Scx, Tnmd, Col1a1, Col1a2 and Tgfb2 genes were all significantly decreased in reduced movement conditions compared to controls during the healing process, following tendon injury.
Fig 4.
EGR1 forced expression in tendons prevents the diminution of tendon gene expression during tendon healing in reduced mechanical load.
(A) Description of the experimental design for sonoporation. 10 μg of EGR1 encoding plasmid and 3.75 μl of MicroMarker microbubbles were injected in the Achilles tendon sheath. Tendons were then stimulated by ultrasound at 1 MHz during 10 minutes at 200 kPa, 40% duty cycle and 10 kHz pulse repeating frequency. The day after EGR1 sonoporation, a surgical lesion of the Achilles tendon was performed followed by a Botox or physiological saline solution injection in the muscle. Two weeks after treatment, tendons were harvested for analyses by RT-q-PCR. (B) RT-q-PCR analysis of tendon gene expression in EGR1-sonoporated tendons versus control-tendons, following tendon injury in immobilization conditions. The mRNA levels of control tendons following injury and Botox injection in muscles were normalized to 1. The error bars represent standard errors of the mean of 6 biological samples of injured tendons of Botox-injected legs in the absence of EGR1 and 10 biological samples of injured tendons of Botox-injected legs in presence of ectopic EGR1. The p values were calculated using the Mann-Whitney test. The mRNA levels of Egr1, Scx, Tnmd, Col1a2 and Tgfb2 were increased in Egr1-sonoporated tendons compared to control tendons, following tendon injury and Botox injection in muscles.
Fig 5.
Schematic representation of EGR1 regulation and function downstream of mechanical signals.
Egr1 expression is regulated by mechanical signals in tendon cells. EGR1 positively regulates the transcription of tendon genes including Scx, Tnmd, Col1a1 and Co1a2. The transcription of Tgfb2 is also regulated by EGR1.