Activated Effects Of Parathyroid Hormone-related Protein On Human Hepatic Stellate Cells

BackgroundParathyroid hormone-related protein (PTHrP) was initially identified from cancers that caused lethal paraneoplastic humoral hypercalcemia. The full biological activity of PTHrP contained within the first34amino acids sequence. PTHrP can act in endocrine, paracrine, and intracrine modes, but especially in paracrine manner. There are three identified action of PTHrP act in an endocrine form manner:(1) the humoral hypercalcemic syndrome, in which PTHrP is produced by tumors and circulates to the bone to stimulate bone resorption;(2) lactation, in which PTHrP is made in the breast and reaches the circulation; and (3) fetal life, where PTHrP regulates maternal-to-fetal placental calcium transport. PTHrP have a similar structure with Parathyroid hormone (PTH) of a N-terminal amino acid sequence homology. PTHrP and its receptor PTHR it is normally produced in every tissues/organs of the body, including liver. Involving many physiological processes in cartilage, vascular smooth muscle, bone, mammary gland development, tooth eruption, pancreas, and others not depicted. PTHrP can produce various effects in different species and tissues with different receptors.The studies of PTHrP are mainly focus on bones and tumors since it was identified. It was already proved that PTHrP is a bone formation promoter in experiments and clinical. Based on the good outcomes, it seems to be an excellent osteogenetic promoter with fast effect and less adverse reaction after years of clinical treatments of osteoporosis. Recently found to occur for PTHrP, it can promote fibrogenesis in the obstructed mouse kidney, with the cooperating of TGF-β1(transforming growth factor-β1), EGF (endothelial growth factor), and VEGF (vascular endothelial growth factor). TGF-β1is a powerful fibrosis promoter and plays a central role in many fibrosis processes, including liver fibrosis. In fact, what effect of PTHrP acts in liver is not yet been estimated.The fibers’formation and degradation result in excessive collagen deposition in hepatic fibrosis, it usually accompany with chronic inflammation and develop to cirrhosis or cancer. The Liver stromal cells related to hepatic fibrosis include hepatic stellate cells (HSCs, previously known as Ito cells, lipocytes, or fat storing cells), Kupper cells and Hepatic portal fibroblasts. During chronic liver fibrogenesis, hepatic stellate cells (HSCs) are a principal fibrogenic cell type that contributes to collagen accumulation. It is well known that hepatocytes release ROS (reactive oxygen species) and some chemokine that can accumulate lymphocytes and macrophages in liver injury. These cells would secrete many solubility cytokine, such as TGF-β1, TNF-α (tumor necrosis factor-α), FGF (fibroblast growth factor). All these cytokine can active HSC into myfibroblasts. Activation of HSCs is a key event in hepatic fibrosis, it acquire contractility and result in extracellular matrix (ECM) when transform to myofibroblasts-like cells. The initiation and persistent of HSCs activation is regulated by many signaling molecules including, and in particular, transforming growth factor-β1(TGF-β1). TGF-β1is the major fibrogenic cytokine in liver disease. HSCs activation can strongly produce TGF-β1to maintain its elevated level and form a positive feedback, then TGF-β1active and recruit more myofibroblasts to injured liver. These activated cells express a-smooth muscle actin (a-SMA), a myofibroblast marker, and synthesize the major composition of ECM fibrillar collagens, the degradation factor MMP (matrix metalloproteinase). The persistence resulting in an attenuated deposition of collagens into the interstitial spaces which finally impairs liver.PTHrP is express in normal liver, it was reported that PTHrP gene expression was induced in rat’s vital organs in1993, including liver, spleen, heart, lung, and kidney in response with injection of LPS (lipopolysaccharide). Hepatic PTHrP messenger RNA (mRNA) levels were induced acutely in rat liver in response to a near lethal dose of endotoxin (LPS), and its protein production were also markedly induced in periportal hepatocytes and caused hepatic acute phase response. These data suggest PTHrP might act as an additional cytokine in liver disease. The hypothesis of the importance of endotoxins in liver damage was first published in1975, now it was already established that endotoxin as a critical cofactor in acute and chronic liver disease in both experimental and clinical, and correlated with the severity of the disease. But what exactly effects of PTHrP in liver cells is not yet evaluated, including HSCs. However, another vivo study of plasma concentrations of PTHrP do not increase in the presence of, or with the severity of hepatic cirrhosis. The concentrations of PTHrP even under the detectable range. It is well known that PTHrP can act in endocrine, paracrine, and intracrine modes, but especially in paracrine manner. Although plasma PTHrP did not significant raise in hepatic cirrhosis. We can not conclude the hypothesis that PTHrP act in a paracrine manner in liver. Thus elucidation of this cytokine in liver awaits further study.Medicine treatments for Osteoporosis (OP) include anti-resorptive agents and bone-forming agents, but the outcomes of OP cause by chronic liver disease is unsatisfied. PTHrP only selectively stimulate bone formation, have less adverse reaction and fast effects in treating Osteoporosis by both experiment and clinical. It is a well accepted and potential choose for OP. But it could promote fibrogenesis in obstructed mouse kidney, and the function is undetermined in liver. Also it was induced in rats in response with injection of LPS and caused hepatic acute phase response, yet its mechanism is not clear. Systemic studies of PTHrP in liver is unavailable. PTHrP as therapy of OP need more lab evident based on the rarely data in liver, so the effects of PTHrP in human liver need more studies.ObjectivesThe activation of hepatic stellate cells has been a popular issue in hepatic fibrosis. The current understand of PTHrP offered a new assessment in this area. It can provide pharmacological evident in treatment of OP, and figure out the pathological mechanism of PTHrP in hepatic cells injury.Methods1. We bought two commercial cell lines, the normal human hepatic stellate cells (HSC) and its activated form LX-2. We examined what effects PTHrP (1-36) might act in them, treated with different concentration(0.1nM、1nM、10nM.100nM) of PTHrP for different time period(6h、12h、24h.48h). After that the total protein, total RNA and cell culture-mediums were collected.2. We tested mRNA expression and protein level by qPCR and Western-blot. Detected the following factor that related to activated:α-SMA、MMP-2、collagen1、TGF-β1.3.Immunofluorescence was performed to identify the expression of α-SMA in HSC and LX-2treated with PTHrP (1-36)(100nM,48h)4. ELISA was used to test the concentrations of TGF-β1in cell culture mediums of HSC after treated PTHrP (1-36)(10-100nM,24-48h)5. Data were summarized as mean±standard error of the mean (S.E.M.) based on experiments repeated in triplicate. Multiple comparisons were analyzed using one-way analysis of variance (ANOVA) with Statistical Package for the Social Sciences (SPSS)13.0software (Chicago, IL). A probability (p)-values less than0.05were considered statistically significant. The results of control group were set to100in Western-blot, and1in qPCR respectively.Results1. After treated with PTHrP (1-36) in various concentrations (0.1-100nM) for different time periods (6-48h), the a-SMA protein (43Kd)(by western-blot) and mRNA (by q-PCR) levels of a-SMA are increased at10-100nM for24-48h in both types of cells, with2-to3-folds (P<0.05). After PTHrP stimulated, western-blot analysis of whole cell lysates demonstrated MMP-2protein (72Kd) increase in both HSCs and LX-2cells. Also PTHrP treatment of HSCs and LX-2cells increased expression of MMP-2mRNA by2-to3-folds, as assessed by comparative real time PCR. In HSCs, in response to PTHrP (1-36) at10-100nM for24-48h, the collagen I protein (138Kd) was significant promoted, as assessed by qPCR mRNA levels caused a2-to3-folds increase (p<0.05) in expression of collagen I as compared to non-treated control cells. In LX-2, exposure of PTHrP (1-36) at10-100nM for48h, collagen I protein and mRNA levels was stimulated, while treated with PTHrP for24h at100nM made these statistically significant.2. To explore the possible effects of PTHrP in HSCs, we observed an increase immunostaining for α-SMA. Incubation with PTHrP(1-36) at100nM for48h, HSCs stained strongly for α-SMA whereas this pattern of staining was absent in control, and LX-2stained significant increased compared with control.3. We found that TGF-(31protein and mRNA were stimulated by PTHrP(1-36) in HSCs at10-100nM concentrations as early as24h, the relative expression of total mRNA and protein are2.963±0.337and356.608±6.587respectively. But LX-2cells is at100nM for48h. In ELISA, as expected, this factor was elicited by PTHrP(1-36) at100nM for48h in HSC of513.6425±82.9245pg/ml,while the control group is190.0733±11.642pg/ml.ConclusionIn summary, we show here for the first time the activated effects,of PTHrP in HSCs, role of the TGF-β1system as a mediator of the profibrogenic action. And its profibrogenic actions in activated stellate cells LX-2. In any event, the present findings further support the notion that PTHrP might act as an additional cytokine in liver disease. And assessing the relative mechanisms to the profibrogenic actions of PTHrP in this setting awaits further study.