Hh signaling pathway plays an essential role in the
Hh signaling pathway plays an essential role in the regulation of invertebrate and vertebrate tissue development and metabolic homeostasis. Components of Hh signaling pathway have been identified in succession, including Hh ligands, membrane protein receptors, cytoplasmic components and transcription factors, all of them are essential for the transduction of Hh signaling . In this pathway, Hh ligands bind to its receptor, Patched1, and reverse inhibition of Smoothened (Smo), a seven-transmembrane protein which has similar sequence with G-protein coupled receptors. Once Smo is reversed repression, the level of its phosphorylation form elevate at the surface of the cell, which further activate transcription factor Gli2 to translocate into nucleus and participate in the activation of target genes, including Gli1, Patched1, Hhip, Cyclin D1 and Bcl2 . In the absence of Hh ligands, Gli2 is phosphorylated and cleaved into shortened peptides that inhibit transcription of target genes. Among the Gli family of transcription factors, Gli1 and Gli2 are Hh-dependent activators whereas Gli3 is an Hh-dependent repressor [14,16]. Nowadays, most of Hh signaling pathway members have been implicated in the development of adipose tissue. Using genome-wide RNAi screening, 500 candidate obesity genes were selected in adult Drosophila, most of them were enriched in Hh signaling . Shh, secreted by hair follicle transit-amplifying PSB 0777 ammonium salt (HF-TACs), could stimulate dermal adipogenesis and hair follicle growth . Sufu (Suppressor of Fused) is an endogenous inhibitor of Hh signaling, adipocyte specific aP2-Sufu KO resulted in near total loss of white adipose tissue, but not brown adipose tissue in mice . Patched1 is a membrane-bound receptor of Hh signaling, deletion of carboxyl-terminal cytoplasmic region of patched1 caused reduction of epididymal adipocyte cell size and total white fat mass . However, all of genetic studies of Hh signaling molecules were not involved in Hhip until now, it is unclear whether Hhip could influence adipogenesis or not.
Material and methods
Discussion Obesity occurs due to excessive lipid deposition, which was caused by increased size of individual adipocytes (hypertrophy) and/or increased numbers of adipocytes (hyperplasia) under appropriate stimuli . It has been reported Hhip was upregulated in glomerular endothelial cells of adult T2D db/db mouse kidneys , however its role in obesity was still unclear. Our current study found that Hhip played a significant role in proliferation and differentiation of preadipocytes. Specially, Hhip promoted adipocytes differentiation via canonical Hh signaling, however, it inhibited preadipocytes proliferation by suppressing cell replication. Overall, our studies could enrich the network of Hh pathway regulating lipid deposition. Formation of adipose tissue is determined by the proliferation and differentiation of adipocytes. Several studies have shown that Hhip could promote cancer cell proliferation and acted as a potential tumor-suppressing modulator. Over-expression of Hhip could significantly decrease the Hhip promoter methylation, which lead to inhibition of gastric cancer cell survival, proliferation, migration and invasion . The heterozygous deficiency of Hhip in mice caused increased Hh activity in tumor, then promoted the secretion of vascular endothelial growth factor (VEGF) by fibroblasts, which displayed a paracrine effect on proliferation of endothelial cell and raised the vascular density in tumors . In our investigation, Hhip could inhibit proliferation of porcine subcutaneous preadipocytes, which was highly consistent with other species and cell types. We speculated there were two reasons for this phenomenon. Firstly, it was determined by the structural characteristics of Hhip. The crystal structure of Hhip and Shh indicated the critical loop from Hhip could bind to the pseudo-active site groove of Shh and directly coordinated its Zn2+ cation . The function of protein is determined by its structure, Hhip is highly conserved across species from yeast to human, therefore, it showed inhibition of proliferation among various cell types. Secondly, several evidences explicitly demonstrated pivotal roles of Hh signaling pathway in activating cell proliferation, Laser irradiation could promote proliferation of mouse pre-osteoblast cell line MC3T3-E1 through activating Hh signaling . Whereas Hhip has been proven to be an endogenous inhibitor of Hh pathway , hence, it inhibited proliferation of porcine subcutaneous preadipocytes. Cellular proliferation is mainly regulated by the cell cycle, which has been divided into four distinct sequential phases (G0/G1, S, G2 and M phases) . A previous research found that in the vertebrate retina, Hh could modulate cell cycle kinetics by activating cyclin D1, cyclin A2, cyclin B1, and cdc25C . In this study, rHhip decreased the expression levels of Cyclin B, Cyclin D and Cyclin E, and attenuated the number of preadipocytes in S phage.