| Cyclin dependent kinase 5（CDK5）regulatory subunit-associated protein 3（CDK5RAP3,also known as C53/LZAP）,which is one of the substrates of ubiquitinated E3 ligase.CDK5RAP3 is widely expressed in tissues and organs throughout the body,and is involved in developmental regulation and tumor formation through ER stress and unfolded protein response（UPR）pathways.Mammary gland is all mammals have,in order to produce milk feeding offspring of glands.Milk synthesis occurs in clusters of differentiated mammary epithelial cells.There are many factors affecting milk yield of dairy cows,and heat stress is one of the most important reasons.Heat Stress（HS）is a buildup of body heat generated either internally by muscle use or externally by the environment.Under non-stress conditions,HSF1 exists primarily as an inactive monomer located throughout the nucleus and the cytoplasm.In its monomeric form,HSF1 activation is repressed by interaction with chaperones such as heat shock proteins HSP70 and HSP90,and TRi C/CCT.In the event of proteotoxic stress such as heat shock,these chaperones are released from HSF1 to perform their protein-folding roles;simultaneously,the export of HSF1 to the cytoplasm is inhibited.These actions allow HSF1 to trimerize and accumulate in the nucleus to stimulate transcription of target genes.Heat stress（HS）in hot climates is a major cause that strongly negatively affects milk yield in dairy cattle,leading to immeasurable economic loss.The heat stress response of bovine mammary epithelial cells（BMECs）is one component of the acute systemic response to HS.Gene networks of BMECs respond to environmental heat loads with both intra-and extracellular signals that coordinate cellular and whole-animal metabolism.CDK5RAP3 is a key regulator of cellular stress response.However,it has not been reported whether CDK5RAP3 can protect bovine mammary epithelial cells against damage caused by heat stress,or whether CDK5RAP3 can regulate the transcription of HSPS m RNA by regulating the heat shock transcription factors（HSFs）,thereby exerting heat stress resistance to reduce cell apoptosis.In this study,the effects of CDK5RAP3 on heat stress response in bovine mammary epithelial cells were investigated.CDK5RAP3 was regarded as the most significant regulator of cellular responses against heat stress,which is associated with dysfunctions of the immune system and animal susceptibility to disease.Despite this,little known about how CDK5RAP3 regulates heat stress response.In this study,CDK5RAP3 conditional Knockout（CKO）mice,CDK5RAP3-/-mouse embryo fibroblasts（MEFs）and bovine mammary epithelial cells（BMECs）were used as an in vitro and in vivo model,respectively to reveal the role of CDK5RAP3 in regulating the heat stress response.The deletion of CDK5RAP3unexpectedly caused animal lethality after 1.5-h heat stimulations.Furthermore,BMECs were re-cultured for eight hours after heat stress and was found that the expression of CDK5RAP3 and HSPs showed a similar fluctuating pattern of increase（0-2,4-6 h）and decrease（2-4,6-8 h）.In addition to the remarkably enhanced expression of heat shock protein,apoptosis rate and endoplasmic reticulum stress,the deletion of CDK5RAP3 also affected nucleoplasmic translocation and trimer formation of heat shock factor 1（HSF1）.These programs were further confirmed in the mammary gland of CDK5RAP3 CKO mice and CDK5RAP3-/-MEFs as well.Interestingly,genetic silencing of HSF1 downregulated CDK5RAP3 expression in BMECs.Immunostaining and immunoprecipitation studies suggested a physical interaction between CDK5RAP3 and HSF1 being co-localized in the cytoplasm and nucleus.Besides,CDK5RAP3 also interacted with HSP90,suggesting an operative machinery at both transcriptional level and protein functionality of HSP90 per se.Together,our findings suggested that CDK5RAP3 works like a novel nucleoplasmic shuttle or molecular chaperone,deeply participating in HSF1-mediated heat stress response and protecting BMECs from heat injury. |
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