CKIP-1Negatively Regulates The Adipogenic Differentiation Of Mesenchymal Stem Cells

Mesoderm-derived mesenchymal stem cells (MSCs) are present in many tissuespostnatally and can differentiate into osteocytes, adipocytes and chondrocytes underappropriate conditions. Aside from their multiple differentiation capacity, MSCs alsoplay important roles in supporting hematopoiesis and in suppressing theimmunoresponse of T lymphocytes. Because of these characteristics, the applicationof MSCs in cytotherapy is promising, such as their use in tissue repair and in reducingimmunological rejection.In present world, improved knowledge of all aspects of adipose biology will berequired to counter the burgeoning epidemic of obesity and relevant metabolicdiseases, which has closed association with abnormal development and function ofadipocytes. The adipocytes constituting white fat tissue are originated from MSCs,which differentiate in response to a series of cues. The extracellular signals aretransduced into nuclear transcription factors via cascades reactions to transcribe manyhundreds of genes, which responsible for establishing the mature fat cell shape andfunction. Transcriptional regulation of adipogenic differentiation is a tightlycontrolled process that is regulated by an elaborate network of transcription factors,cofactors and signalling intermediates from numerous pathways.Among these transcription factors, the most notable are CCAAT/enhancer bindingproteins(C/EBP) and the peroxisome proliferator-activated receptors gamma(PPAR). Actually, it is well accepted that both C/EBPα and PPARγ act as criticalregulators of adipogenesis, since deficiency of either of them shows thedevelopmental defects of white adipose in mouse model. Consequently, in our effortsto gain a complete understanding of the processes regulating the function ofadipocytes, it is important to identify the mechanisms regulating transcription of theseadipogenic transcription factors.CKIP-1is a PH domain containing protein, involving in apoptosis, cell skeletonmaintaining and so on. In recent years, it was found CKIP-1promoted E3ligaseSmurf1's activities and negatively regulated osteogenesis in mouse model. It is wellknown that osteoblasts and adipoblasts are descended from common progenitors-- MSCs. Yet, the function of CKIP-1in the adipogenic differentiation of MSCs is notknown. In the present study, we isolated and richened MSCs from murine compactbone of CKIP-1wild-type (WT) and knockout (KO) mice, and further identified theirpurity and CFU-F capacity. In the subsequent multiple differentiation assays, wefound that MSCs lacking CKIP-1display enhanced adipogenesis after exposed tostimuli than their wild-type counterparts, and increased transcriptional levels of thecritical adipogenic marker genes, especially for C/EBPα. Due to the HDAC1represscomplex is curial for the transcription of C/EBPα, we investigated the association ofCKIP-1and HDAC1in adipogenesis of MSCs. It is found that CKIP-1can localize inthe nucleus and interact with HDAC1directly. This is the frist time that CKIP-1hasbeen observed in the nucleus under physiological conditions. Our date also show thatHDAC1constitutively binds the promoter of C/EBPα in MSCs, but dislodges duringthe early phase of adipogenesis. In the presence of CKIP-1, the dislodgment ofHDAC1from the promoter of C/EBPα is weakened, which suppresses thetranscription of C/EBPα and adipogenesis. Furthermore, CKIP-1deficiency leadsmore Ac-histone3and RNA pol II in the promoter of C/EBPα during adipogenesis ofMSCs, but less HDAC1. Moreover, on high fat diet, CKIP-1KO mice showaccelerated body weight gain, increased total fat mass and severe fatty liverphenotype than their WT counterparts. Taken together, we conclude that CKIP-1is anovel negative regulator of adipogenesis of MSCs.In addition, we observed some interesting phenomenons by chance. MSCs culturewas first developed by Friedenstein et al. from bone marrow, and since then, bonemarrow has become the common source for isolating MSCs. Thus far, MSCs havebeen isolated from the bone marrow of many species, including humans, rats and pigs.In contrast to other species, murine MSCs cannot be easily harvested from the bonemarrow due to contamination by hematopoietic cells, and this contamination isdifficult to eliminate using the characteristic plastic adherence of MSCs. Recentstudies showed that the compact bone tissue of long bones is a novel source of MSCsfor both humans and mice. These studies developed a novel method to obtainhigh-purity murine MSCs by culturing collagenase-digested compact bone fragments.Some procedures in this method help avoid hematopoietic cell contamination, such asby removing bone marrow before collagenase digestion and discarding the releasedcells after collagenase digestion. Furthermore, subsequent experiments showed thatcompact bone is a richer source of MSCs than the marrow plug within it. Based on these findings, we postulated that murine MSCs could be isolated from the compactbone of the calvaria, which has a relatively low bone marrow content. In this study,we found that cells migrating from the calvaria possess morphological characteristicsand surface antigen profiles similar to those of MSCs derived from long bones.However, these calvaria-derived cells highly expressed the osteogenic transcriptionfactor osterix. The calvaria-derived cells lost their adipogenic capacity but gained ahigher osteogenic capacity. These results suggest that not all types of murine compactbones in the body are sources of MSCs and that the differentiation fate ofmesenchymal stem/progenitor cells in different types of compact bones is alreadycommitted. The cells that migrate from the calvaria should be considered progenitorcells rather than MSCs.