Expression Of FSHR In Chondrocytes And The Effect Of FSH On Chondrocytes

Backgrounds:Cartilage tissue consist of chondrocytes,matrix and fibers,chondrocytes are the only cells in cartilage tissue,and cartilage tissue are divided into cartilagines vera,elastic cartilage,fibrocartilagines according to the fiber composition.Hyaline cartilage is mainly distributed throughout articular cartilage and is widely distributed in vivo.Articular cartilage consists of dense connective tissue collagen fibers to form the basic framework and chondrocytes distributed in them.Chondrocytes maintain normal metabolism of articular cartilage.Joint movement plays an important role in maintaining the normal structure of articular cartilage.Articular cartilage is not only smooth but also elastic and can be absorbed to the maximum extent.Buffer stress.After articular cartilage injury,the force absorption is reduced,joint injury and degeneration will be gradually aggravated.Follicle stimulating hormone(FSH)is one of the glycoprotein hormones secreted by the pituitary gland and is a key signal molecule in the hypothalamus-pituitary-endocrine axis.FSH acts mainly by binding with FSHR.It is generally believed that pituitary glycoprotein hormone receptors exist only in specific endocrine target organs and tissues.The distribution of pituitary glycoprotein hormone receptors in extraendocrine tissues is recently reported and controversial.In non-classical tissues,glucoprotein hormone receptor content is usually low,and it is only expressed in a specific stage of development(such as tumorigenesis or tissue repair).For example,hormone synthesis is inhibited(as in pituitary failure)or hormone synthesis increases sharply(hyperhypophysis due to endocrine failure).These peripheral non-classical glycoprotein hormone receptors are activated and play a biological role.In women,follicle stimulating hormone receptor(FSHR)is specifically expressed on the membrane surface of ovarian granulosa cells.Activation of intracellular signal transduction ultimately determines the proliferation of granulosa cells.The high expression of FSHR in the ovary plays a key role in follicle development and maturation.FSHR is specifically expressed in Sertoli cells of testis and regulates spermatogenic function by promoting spermatogenesis.It is found in the kidney and liver of zebrafish.In humans,the FSHR expression has been reported to be a novel approach to the regulation of FSH-FSHR signal in adipose tissue.It has opened up a new research field of pituitary glycoprotein receptor.With the deepening of various studies,people had previously thought that endocrine regulation was carried out through various functional axes,and recognized that the level of hormone action remained at the overall level or at the level of tissues and organs.The hormone secretion system and the diffuse hormone secretion system.These hormones include peptides,amines,and other substances that reach the various parts of the body's environment through a variety of forms,such as endocrine,autocrine,paracrine,etc.play a stabilizing role and provide fine regulation.Osteoarthritis(OA)is the most common osteoarticular disease in the middle and old age.Studies have shown that the most early pathological manifestation of OA is the destruction of articular cartilage matrix.The disease is characterized by loss of cartilage and changes in bone structure,including marginal growth,osteophytes and sclerosis.Osteoarthritis also includes other soft tissue structures,including muscle,tendon and ligament weakness,and symptomatic synovitis.The incidence of postmenopausal women was significantly higher than that of men,and the incidence of low estrogen level in women increased further.FSH stimulated follicle secretion of estrogen.It is also regulated by negative feedback of estrogen.When gonadal function is decreased such as menopause,ovarian failure,estrogen level plummet and FSH level rises.It is also considered as a diagnostic marker of menopause.However,during the perimenopausal period,the level of FSH in women was significantly increased when the estrogen level was in the normal range.We assume that the rapid increase of FSH during perimenopausal and menopause may be directly involved in regulating the proliferation and differentiation of chondrocytes.Current studies have shown that FSHR is not only expressed on the surface of ovarian tissue,but also widely expressed in non-ovarian tissue cells,such as adipocytes,fibroblasts,lymphocytes.At the same time,chondrocytes also express a variety of hormone receptors,such as growth hormone receptor,insulin-like growth factor-1,glucocorticoid receptor,thyroid hormone receptor,estrogen receptor.Androgen receptor,vitamin D receptor,leptin receptor,etc.The growth and differentiation of chondrocytes are regulated directly or indirectly by many hormones.Thyroid hormone and growth hormone can promote the growth and maturation of the epiphyseal plateWhether chondrocytes express FSHRs has not been studied.No study has been focused on FSH and chondrocyte differentiation and metabolism.We are pleased to find the presence of FSHR in human cartilage tissue,which is the first time in the world that FSHR has been found in cartilage tissue.The expression and function of FSHR in chondrocytes were discussed for the first time,and the viewpoint that FSH directly regulated the differentiation and metabolism of chondrocytes was put forward for the first time.The regulation of FSH-FSHR signal opens up a new research direction of postmenopausal osteoarthritis and opens up a new research field of pituitary glycoprotein receptor.Objectives:1.To investigate the expression of FSHR in chondrocytes;To observe the expression of FSHR mRNA and protein in normal human cartilage tissue and mouse chondrocytes cultured in vitro.And the changes of cAMP content in chondrocytes after forskolin stimulation in primary chondrocytes of mice to determine whether the FSHR on the surface of chondrocytes has active function.2.To investigate the mechanism of FSH acting on chondrocytes.Methods:1.Collection of human normal articular cartilage tissue and human ovarian tissues:Human cartilage tissue was obtained from patients who received knee joint replacement surgery because of severe osteoarthritis in the Department of Orthopedics' ward of Shandong Provincial Hospital.Human ovarian tissue was obtained from patients who had undergone oophorectomy in the gynecology ward of Shandong Provincial Hospital due to a malignant ovarian tumor.2.Mouse chondrocytes were cultured with 10%fetal bovine serum,100 IU/ml penicillin and 100 p g/ml streptomycin.When the chondrocytes grew to 80%?85%in the culture dish,FSH was added.Before the treatment,the original complete culture medium was discarded and the culture dish was washed twice with PBS.The basic culture medium was cultured overnight and then the corresponding treatment was added.3.Measure of FSHR in chondrocytes:Mouse primary chondrocytes and human articular cartilage tissues were examined.The expression and sequence of FSHR mRNA by reverse transcription polymerase chain reaction(RT-PCR)and sequenced,respectively,and its protein expression was tested using western blotting and location was observed under immunofluorescence microscopy.Ovarian tissue was as a positive control.4.To determine whether the FSHR protein observed in chondrocytes was functional,After FSH stimulated mouse chondrocytes,intracellular cAMP levels were assessed by ELISA,and gene expression relative to Mouse WNT Signaling Pathway was tested by RT2 Profiler PCR Arrays.Results:1.FSHR mRNA is expressed in primary mouse chondrocytes and human articular cartilage,respectively.According to the difference regions of mouse and human FSH receptor(FSHR),three pairs of primers were designed and representative extramembrane(extra),transmembrane(trans),and intramembrane(intra)of the FSHR,respectively.The cDNA was from ovary tissues as a positive control while was substituted by double distilled water as the negative control,respectively.The PCR products of FSHR from mouse chondrocytes and human articular cartilage tissue were sequenced to assess the specificity of the amplified fragments.The results showed that the FSHR sequences of mouse chondrocytes were identical to the published mRNA sequence of mouse ovarian tissue.The results of human cartilage tissue FSHR sequences were identical to the published mRNA sequence of human FSHR derived from ovarian tissue.2.Western blotting analysis of FSHR proteins in primary mouse chondrocytes and human cartilage:The proteins of mouse and human samples appeared respectively at 78 kDa with a band representing FSHR as well as the positive control of corresponding ovary tissues.Immunofluorescence was used to examine the cellular distribution of FSHR protein in mouse chondrocytes.3.Functional analysis of chondrocyte FSHR:cAMP reactivity of chondrocytes to FSH.Primary mouse chondrocytes isolated from newborn mice were seeded on monolayer.The chondrocytes were stimulated with FSH(5,10,20 or 40 ng/ml)and forskolin(100 mM)for 5 min,respectively.Cell lysates were collected to detect cAMP contents.The total proteins of corresponding each well normalized to cAMP contents.Forskolin,as a positive control,evidently elevates the cAMP level(P<0.001),but FSH does not change the cAMP content in chondrocytes compared to vehicle treatment(IBMX/100 mM).A quantitative gene array identifying WNT signaling pathway genes was associated with test group FSH factor.There were three genes over-expressed,namely Fos-like antigen 1(Fosll),Ras homolog gene family,member U(Rhou),and Dickkopf homolog 1(Dkk1).Conclusions and Significance:In this study,the expression and sequence of FSHR mRNA by reverse transcription polymerase chain reaction(RT-PCR)and sequenced,respectively,and its protein expression was tested using western blotting and location was observed under immunofluorescence microscopy.Ovarian tissue was as a positive control.In our experiments,the results showed that FSH failed to significantly stimulate cAMP production in mouse primary chondrocytes at different concentrations.To further verify the functionality of FSHR,we conducted a PCR-array test.Three up-regulated genes were screened out by Wnt 0-catenin gene pathway,namely Fos-like antigen 1(Fosl1),Ras homolog gene family,member U(Rhou),and Dickkopf homolog 1(Dkk1).The discovery of functional FSHR on the surface of chondrocytes will provide a new research direction for the potential regulation of FSH in cartilage physiological function and pathophysiological process.It provides new research ideas and therapeutic targets for exploring the relationship between ovarian dysfunction and cartilage function change,and has important theoretical value and application prospect.