P21 Deficiency Delays Regeneration of Skeletal Muscular Tissue

<div><p>The potential relationship between cell cycle checkpoint control and tissue regeneration has been indicated. Despite considerable research being focused on the relationship between p21 and myogenesis, p21 function in skeletal muscle regeneration remains unclear. To clarify this, muscle injury model was recreated by intramuscular injection of bupivacaine hydrochloride in the soleus of p21 knockout (KO) mice and wild type (WT) mice. The mice were sacrificed at 3, 14, and 28 days post-operation. The results of hematoxylin-eosin staining and immunofluorescence of muscle membrane indicated that muscle regeneration was delayed in p21 KO mice. <i>Cyclin D1</i> mRNA expression and both Ki-67 and PCNA immunohistochemistry suggested that p21 deficiency increased cell cycle and muscle cell proliferation. F4/80 immunohistochemistry also suggested the increase of immune response in p21 KO mice. On the other hand, both the mRNA expression and western blot analysis of <i>MyoD</i>, <i>myogenin</i>, and <i>Pax7</i> indicated that muscular differentiation was delayed in p21KO mice. Considering these results, we confirmed that muscle injury causes an increase in cell proliferation. However, muscle differentiation in p21 KO mice was inhibited due to the low expression of muscular synthesis genes, leading to a delay in the muscular regeneration. Thus, we conclude that p21 plays an important role in the <i>in vivo</i> healing process in muscular injury.</p></div

Immunofluorescence analysis of muscle basement membrane and plasma membrane.

<p>The expression of DAPI, laminin, and dystrophin was examined in wild-type (WT; upper panel) and p21 knockout (KO; lower panel) mice. (A) Expression in control, (B) Expression in 3 days, (C) Expression in 14 days, and (D) Expression in 28 days post-operation. (Scale bar = 50μm). (E) Quantification of laminin expression, (F) Quantification of dystrophin expression. (A-F) The injured membrane structure was almost repaired 14 days after injury in WT group, but not in the p21 KO group (p < 0.05).</p

Comparison of muscle weight/body weight.

<p>± Standard error.</p><p>Comparison of muscle weight/body weight.</p

Western blot analysis of myogenic differentiation-related proteins.

<p>WT: wild type, KO: knockout. The expression levels of <i>MyoD</i>, <i>myogenin</i>, and <i>Pax7</i> was increased 3 days after injury in WT mice, but not in p21KO mice.</p

Additional file 1: of p21 deficiency is susceptible to osteoarthritis through STAT3 phosphorylation

The primer sequence for detection of human p21, COL2A1, ACAN, MMP-3, MMP-13, ADAMTS-4, ADAMTS-5. (TIFF 58 kb

Ki-67 expression after muscular injury.

<p>(A) Immunohistochemistry of Ki-67, (B) Quantitative analysis of Ki-67. (A and B) Ki-67 expression levels in WT and p21KO mice were significantly different at control and 3 days after injury (p < 0.05). Ki-67 expression in p21KO mice at 3 days after injury was the highest (p < 0.05).</p

Histological analysis of muscle tissues control, 3, 14, and 28 days post-operation hematoxylin-eosin (HE) staining.

<p>WT: wild type, KO: knockout. (A) Muscle tissues at control and 3 days post-operation, (B) Muscle tissues at 14 and 28 days post-operation. (Scale bar = 50μm). (A and B) The injured muscle was almost recovered 14 days after injury in WT group, but not in the p21 KO group.</p

PCNA and F4/80 immunohistochemical expression after muscular injury.

<p>WT: wild type, KO: knockout. (A) Immunohistochemistry of PCNA. (Scale bar = 50μm), (B) Quantitative analysis of PCNA-positive cells, (C) Immunohistochemistry of F4/80. (Scale bar = 50μm), (D) Quantitative analysis of F4/80-positive cells. (B and D) Both PCNA and F4/80 expression in p21KO mice at 3 days after injury were the highest (p < 0.05).</p

Relative expression of <i>Cyclin D1</i> mRNA (expressed in the logarithmic scale).

<p>WT: wild type, KO: knockout. <i>Cyclin D1</i> expression levels were significantly higher in p21 KO group than in WT group, through the 28 days post-injury.</p

mRNA expression profiles of myogenic markers.

<p>The relative mRNA expression of (A) <i>MyoD</i>, (B) <i>myogenin</i>, and (C) <i>Pax7</i> was determined in wild-type (WT) and p21 knockout (KO) mice using the relative gene expression level at 0 day post-operation as control. (A-C) The mRNA expression of <i>MyoD</i>, <i>myogenin</i>, and <i>Pax7</i> peaked on day 3 in WT mice and on day 14 for <i>MyoD</i> and <i>myogenin</i> in KO mice. The peaks for WT were observed to be stronger than those for KO mice.</p