Molecular Cloning, Characterization And Primary Functional Analysis Of HSPC016 In Dermal Papilla Cells

Hair has many useful biological functions, such as to protect the organism from heat loss and afford the underlying epidermis a "first line of defense" from abrasion and penetration of noxious chemical agents, etc. As for humankinds, the most important function of hair is to form varied size of body hairs, such as eyelashes, eyebrows, scalp hairs and so on, and these body hairs act as physical medium of social communication; In fact, scalp, facial, and body hairs are essentially the only body part by which an individual can shape to influence social relationship. Patients with hair loss such as alopecia or excessive hair growth often suffer tremendously. Depression, low self-confidence, and humiliation are observed frequently in the patients of all ages. Not surprisingly, understanding the morphogenetic mechanism, growth cycle of hair, and developing new drugs to treat hair disease is the key subject in the hair biological research area.Hair follicle, the main body of hair, is a regenerative system. Once hair follicle formed in utero, it starts to cycle continuously over the total lifetime of a mammal including human. Follicle cycling includes anagen, catagen and telogen. If anagen stopped ahead and telogen prolonged abnormally, alopecia will occur. Normal development and cycling of hair follicle depend on the interaction of the follicular epithelium with the adjacent mesenchymal follicle papillae. The dermal papilla cells (DPC) is the unique cell within follicle papillae which induces hair-follicle formation from the overlying epithelium during fetal development, and at the onset of each new follicular cycle in adults DPC play important roles in the control of follicle cycling through interactions with follicle epithelia.Although most of our current knowledge of the substances that modulate hair growth in humans is derived from clinical observations, studies in mice/rat models have identified some of the molecular events associated with hair follicle cycling, such as fibroblast growth factors (FGFs), bone morphogenetic proteins (BMPs), sonic hedgehog (Shh), neurotrophins, platelet derived growth factor (PDGF), and so on . Some of the growth factors or growthfactor receptors are secreted by cell lineages within follicle including DPC, ORS (out root sheath) cells, matrix cells. Further more, as for an individual factor, it is only produced at a given period in the cycling, but stopped at other period. However, it is still unclear that which signal transduction pathway promote or stop these modulatory factors secretion and which specific cells control the above process.Inamatsu et al provided evidence that DPC in the state of aggregative growth pattern in vitro still sustained the potency to induce hair follicles formation from afollicular skin. In order to know which gene expressed in DPC at the state of aggregative growth pattern, Song et al set up the Human DPC subtractive library and screened out some up-regulated genes and down-regulated genes successfully. HSPC016, first identified in CD34+ hematopoietic stem/progenitor cells, is one of the differential expression gene in DPC, but its function is not clear. Since the evidence that DPC have some characteristics of stem cells, such as aggregative growth behavior, some biologists had DPC generated erythroid and myeloid cells in vitro and yield multiple haematopoietic lineages for at least a year when cultured and transplanted into lethally irradiated mice , HSPC016 may play an important role in DPC. We therefore cloned its full length cDNA sequence in DPC by RACE (rapid amplification of cDNAends), predicted its possible function by bioinformatics analysis and verified the function by silencing the mRNA oiHSPC016.In our research, two isoforms of HSPC016 were identified with full length of 400bp and 493bp cDNA (GenBank accession No: AY508979% ). Bioinformatic analysisand database searching on the internet indicated that this gene was mapped on chromosome 3 q21.31 and included an open reading frame with 195bp coding for an expected 64aa soluble protein. #NN=9/23 algorithms on PSORT WWW Server (http://psort.ims. u-tokvo.ac.ip) showed the location probability in nuclear was 56.5% for the protein, 26.1% in cytoplasm and 17.4% in mitochondrion correspondingly. The putative protein belonged to PD053992 family and was homologous to T2FA gene in domain. As we know that T2FA is transcription initiation factor IIF, alpha subunit (TFIDF-alpha) in human. It can promote transcription elongation of other genes. Combined all the analyses above, HSPC0J6 may act as a transcript factor and be involved in protein-protein interactions in one of the pathways about transcriptional regulation.In order to verify the bioinformatic analysis results and observe the growth anddifferentiation of DPC with HSPC0J6 gene knock down, we designed 3 small interfering RNA duplexes (siRNA), siRNAl, siRNA2 and siRNA3, according to the full-length mRNA of HSPC016 gene. These short interfering fragments were inserted into plasmid psilencer-3.1-Hl-hygro respectively, to construct interfering vector psilencer-3.1-Hl-hygro-siRNAl, psilencer-3.1-HI-hygro-siRNA2 and psilencer-3.l-Hl-hygro-siRNA3. Then, we transfected these vectors bearing siRNA into DPC, let the vector synthesize and release siRNA aimed at HSPC016 continuously. Our result indicated that the siRNAs we designed for HSPC016 gene silenced HSPC016 expression in DPC successfully. The HSPC016 knocked down DPC grew much slower than normal DPC. They lost aggregative properties and the shape of cell also changed significantly. The normal DPC were ellipse or egg-like cells, but the HSPC016 negative DPC switched into fibroblast-like cells. Our findings indicated that HSPC016 gene played a critical role in the control of DPC proliferation and differentiation. It laid a solid foundation for further investigation of the molecular mechanism of HSPC016 gene.To sum up, we have identified and characterized HSPC016 gene in DPC. The primary functional analysis using RNAi technology ascertained that HSPC016 was closely related to the proliferation and differentiation of DPC. The gene was very important to sustain the functional condition of DPC. Our research results laid a solid foundation for further studying the molecular mechanism of HSPC016 gene modulation , and for investigating new clinical gene therapy for patients with hair loss.