The Research On Regulating Mechanism Of PTEN On HSFBs Apoptosis And Functions

Background: Keloids and hypertrophic scars (HS) are skin abnormalities that areunique to humans and are characterized by excessive deposition of collagen and invasivegrowth of fibroblasts in the dermis and subcutaneous tissues secondary to traumatic orsurgical injuries. The exuberant scarring of HS results typically in disfigurement anddysfunction, which would bring not only great physical pain, but also psychological stressto the patients. Current treatments of HS, which have their own sideffects and limits, oftenhave an unsatisfactory outcome frustrating both the patient and the clinician alike. Toachieve a successful and effective treatment remains to be the priority on recent researchtechniques.Dermal fibroblasts play a major role in HS formation. As shown in recently study, theiraberrant proliferation, apoptosis inhibition and excessive production of extracellular matrix(ECM) were considerate as main attributers to the pathogenesis of HS formation. Behaviorsof hypertrophic scar fibroblasts (HSFB) are regulated by certain factors in vivo. Previousstudies suggested that some apoptosis related genes, such as fas, p53and bcl-2were keyregulators in HSFB proliferation and apoptosis; however, the mechanisms are still unclearuntil now.Phosphatase and tensin homologue (PTEN) is a negative regulator of AKT signalingand a tumor suppressor that exhibits dual-specific phosphatase activity; it is also a keyregulator of apoptosis, cell cycle and cell motility. The loss of PTEN function because ofdeletion, mutation, methylation, or decreased expression has been found in human cancersand some fibrotic diseases. In idiopathic pulmonary fibrosis and liver fibrosis both mRNAand protein levels of PTEN were suppressed. We therefore hypothesized that PTEN mightbe involved in the PI3K/AKT signal pathway, and further participated in HS pathogenesis.In our previous experiment, PTEN expression in HS tissue was significantly decreasedcompared to normal skin (NS) tissue, whereas p-AKT expression was increased, indicating an inverse correlation between PTEN and p-AKT. Therefore, we conjectured that inabnormal tissue repairment, the loss of down-regulation of PI3K/AKT signal pathwaycaused by PTEN expression decreasing would weaken their inhibiting effect on fibroblastsproliferation, therefore resulted in aberrant fibroblast proliferation, redundant collagensecretion, excessive ECM production, and finally HS formation.Objection: To observe both the protein and mRNA expression of PTEN and AKT inHS and to explore PTEN's regulating effect on the proliferation, apoptosis anddifferentiation of HSFB. These results wound reveal a novel biological role of PTEN in HSpathogenesis and treatment, and provide new insights into the downstream signaltransduction pathway of PTEN.Methods: Cultured fibroblasts were obtained from NS and HS samples and cells at3–6passages were used in the experiments. Immunohistochemical staining was used toobserve the expression of PTEN and p-AKT in both NS and HS tissues. High level ofPTEN expression was then induced in both NSF and HSF using an adenovirus vector.Bpv(pic), a PTEN specific inhibitor, was also used in our experiments. Alterations onproliferation, differentiation and migration were detected and the signal transductionpathways were explored too.Results:1. Measuring by PT-PCR, immunohistochemistry and Western Blot, PTEN expressionin HS tissue was significantly decreased compared to NS tissue, whereas p-AKT expressionwas increased.2. Observed under laser confocal fluorescence microscope, PTEN was stronglyexpressed in NSF, and located mainly in cytoplasm and nucleus, while p-AKT expressionwas weak. On the contrary, PTEN expression was barely seen in HSFB, while p-AKTexpression was positively seen in cytoplasm and nucleus.3. Recombinant mutants were confirmed using PCR and DNA sequencing. High-titeradenovirus was obtained. PTEN expression in fibroblasts after infection was observedunder confocal laser scanning microscope.4. Measured by MTT assay, the proliferation of HSFs was negatively regulated byPTEN expression, while on effect was detected on NSFBs proliferation. On the other hand,bpv(pic) could promote NSFBs proliferation without any effect on HSFBs. 5. The results of Flow Cytometry showed HSFBs were arrested at G0-G1phase afterPTEN expression increased, while no change was found in NSFBs. At the same time, Sphase rate increased after PTEN expression inhibited in NSFBs. The same results couldalso been obtained by immunofluorescence cytochemistry of PCNA.6. Analyzed by TUNEL, apoptosis of HSFBs was significantly increased afterupregulation of PTEN, while no change of apoptosis was found in NSFBs.7. Caspase-3activation was significantly enhanced in HSFBs than in NSFBs afterPTEN up regulation, while no difference was found in either cells when PTEN expressionwas inhibited.8. The synthesis of collagen was determined by measuring the incorporation of3H-proline in fibroblasts. Collagen synthesis in HSFBs was specifically inhibited by PTENupregulation, while PTEN downregulation promoted it in NSFBs. The similar results onprocollagen I and III were obtained by RT-PCR too.9. Upregulation of PTEN expression could significantly inhibit the expression ofTGF-β1in HSFBs.10. PTEN could reverse the trans-differentiation of HSFBs by inhibiting α-SMAexpression.11. Measuring by Western Blot, the cellular content of AKT, c-Raf, MEK1/2, ERK1/2and p38were all decreased in different degrees after PTEN expression increased.Conclusions:1. PTEN expression in HS tissue was significantly decreased compared to NS tissue,whereas p-AKT expression was increased, indicating a negative relationship between PTENand p-AKT.2. Both HSFBs and NSFBs that steadily expressed high level of PTEN weresuccessfully obtained.3. Increasing cellular PTEN expression could effectively inhibit the proliferation ofHSFs and induce their apoptosis.4. PTEN could act on some biological functions of HSFBs, such as inhibiting theircollagen synthesis, reducing TGF-β1production, and decreasing α-SMA expression, andtherefore reverse their transdifferentiation.5. PTEN affects the proliferation and activation of HSFBs through regulating MAPK and AKT signal transduction pathways. The most possible pathway are Ras→Raf→MEK→ERK/p38and PI3K→Akt.