Regulation Of Hoxa5 On Proliferation And Apoptosis Of Adipocytes And Its Molecular Mechanism

Cell proliferation and apoptosis are important basises for the growth and development of organisms.The development and deposition of adipose tissue contain two different processes,the increase of adipocyte number and adipocyte volume.And the excessive deposition of fat causes a unhealthy status,which results form not only energy metabolism disorders,but also the imbalance of adipocyte proliferation and apoptosis.Hoxa5 gene,one of the members in Hox gene family,is an important transcription factor associated with development.Recent studies have found Hox genes regulate the development of white and brown adipose from different parts in human according to an anterior-posterior expression pattern.And the methylation of Hoxa5 regulates the differentiation of adipocytes in different adipose tissues of mice.So,we hypothesized that Hoxa5 may also be involved in regulating the proliferation and apoptosis of adipocytes.However,recent studies of Hoxa5 have focused on cancer cells,and the regulatory mechanism of adipocyte proliferation and apoptosis is unclear.In order to clarify the regulation of Hoxa5 on adipocyte proliferation and apoptosis and its molecular mechanism,we overexpressed or interfered Hoxa5 in adipocytes.Finally,we got the following results:1.Hoxa5 inhibited adipocyte proliferation by increasing cell cycle arrest.We transfected adipocytes with overexpression vector of Hoxa5(pc-Hoxa5)and interference vector of Hoxa5(sh-Hoxa5).Our results showed pc-Hoxa5 significantly reduced adipocyte number with cell counting.EdU staining and PI staining combined with flow cytometry analysis showed adipocyte number in G0/G1 was increased in pc-Hoxa5 group,while in S phase,it was obviously decreased.Further,pc-Hoxa5 was also decreased the content of DNA in nucleus.RT-PCR and Western Blot analysis showed pc-Hoxa5 inhibited PCNA,Cyclin E and Cyclin D,and promoted p27 and p53.In sh-Hoxa5 group,the results were opposite.Therefore,the data indicated that Hoxa5 inhibited adipocyte proliferation by increasing cycle arrest.Further measurements indicated lower phosphorylation of JAK2 and STAT3 accompanied by down-regulated Cyclin E and up-regulated p53 protein.So it revealed Hoxa5 suppressed adipocyte proliferation by inhibiting JAK2/STAT3 signaling pathway.2.Hoxa5 promoted adipocyte apoptosis through mitochondrial pathway.Hoxa5 significantly increased the number of apoptotic cells with Hoechst staining.Annexin V/PI staining and an analysis by flow cytometry demonstrated that a higher percentage of apoptotic cells in early and late stage after pc-Hoxa5 treatment as compared with control group.Hoxa5 increased the fragments of genome DNA in nucleus with TUNEL staining.JC-1 staining and flow cytometry analysis showed pc-Hoxa5 treatment reduced mitochondrial membrane potential.Moreover,pc-Hoxa5 treatment significantly increased Cytochrome-c(Cyt-c)protein.Those changes were correlated with the increase of mitochondrial pro-apoptotic genes Bax,Bid,Bad,Caspase-9,Caspase-3 and the reduction of mitochondrial anti-apoptotic gene Bcl-2 after forcing expression of Hoxa5.In sh-Hoxa5 group,the results were opposite.Our above data collectively revealed Hoxa5 increased adipocyte apoptosis through mitochondrial pathway.Further study found that the Akt /mTORC1/S6k1 pathway was also involved in it.3.Hoxa5 controlled the proliferation and apoptosis of adipocytes by transcriptional regulation.Analysis of our online prediction demonstrated the promoter of Ccne1,a cell cycle-related gene,contained an important binding site,which was a potential target of Hoxa5 protein.And there were also an important binding site in the promoter region of Bax,an apoptosis-related gene.An analysis of dual luciferase reporter system showed Hoxa5 protein negatively regulated Ccne1,and positively regulated Bax.These results further demonstrated that Hoxa5 inhibited adipocyte proliferation by increasing cell cycle arrest and promoted adipocyte apoptosis through mitochondrial pathway.Taken together,in the study,we clarified the effects of Hoxa5 on regulation of adipocyte proliferation and apoptosis.Hoxa5 inhibited adipocyte proliferation by increasing cell cycle arrest,and promoted adipocyte apoptosis through mitochondrial pathway.Also,JAK2/STAT3 and Akt /mTORC1/S6k1 pathway were involved in the process.