| Background:Achondroplasia(ACH)is a cartilage metabolic disease which has been a puzzle for the clinicians for quite a long time.The clinical symptoms are mainly for short limbs and craniofacial deformities,and the length of the spine are often normal.People have continually done the research on the ACH after Oberklaid found the disease since 1979.According to current research data,ACH is an autosomal dominant mutation of a genetic disease,and usually can be found in childhood.The prognosis of ACH is poor and the disease is the result of chromosomal mutation,which lacking of treatment.Besides,cartilage is wide-spreading in human body and as a very important organization,once the developmental was disordered,it will involve multiple systems and organs.By measuring ACH patients multiple radiographic parameters,Oberklaid et al summarized ACH clinical manifestations as follows:the trunk and limbs of a new born baby is disproportionately such as larger head,shorter limbs,however the length of trunk is normal,which can be called short-limb dwarfism type.What‘s more,the proximal limbs are much shorter than the distal limbs.For example,the femur is shorter than the tibia and fibula,while the humerus is shorter than the ulna and radius.This feature is becoming more obvious with aging,gradually forming dwarfism.In addition,the facial feature is bridge of the nose collapsing,jaw protruding and forehead widening.Middle finger and ring finger cannot be folded,appearing a typical "trident hand." Sometimes the elbow is flexion contracture and the radial head is dislocation,accompanying short,cuved,arched limb,and bloated muscles.The length of spine is closed to the normal standard,but it might be a thoracic kyphosis and magnum stenosis in infancy.The main symptom is waist and leg pain,and intermittent claudication.Generally speaking,the intelligence is not affected.In addition,the view of pathogenesis is a lot but not unified.But as we all know that the fibroblast growth factor receptor 3(Fibroblast growth factor receptor,FGFR3)gene mutations can often lead to short-leg type dwarfism.Besides,FGFR1,FGFR2,TWIST1 and Spred-2 gene mutation can also lead to ACH.According to the classical theory,the growth plate in the distal femur is divided into 4 layers,from the distal to the proximal femur include:resting chondrocytes,proliferative chondrocytes,prehypertrophic chondrocytes and hypertrophic chondrocytes.The resting chondrocytes are mainly in the resting state,as a precursor cell of proliferative chondrocytes,mainly secreting SOX9,collagen type 2(Col2),PTC1(Ihh target molecules)and other signal molecules.The proliferative chondrocytes also called columnar cells,which means a large number of cells in proliferative phase,secreting PTC 1,Fgfr3,NKX3.2 signal molecular.While the prehypertrophic chondrocytes are immaturate cells which located between at the end of proliferating chondrocytese and the beginning of the hypertrophic chondrocytes,secreting Ihh,PPR,BMP6,AKP,RUNX2,Osterix(OSX)and other signal molecules.In addition,hypertrophic chondrocytes are mature chondrocytes,mainly secreting Col10 signal molecules.Many people are also definited the zone between hypertrophic chondrocytes and osteoblasts as the terminal hypertrophic chondrocytes.This is the district that chondrocytes degenerate and apoptosis,cartilage matrix become calcification,and eventually osteoblasts are formed which maily secreting osteopontin(OPN).Later osteoblasts participate in the formation of trabecular bone,and cartilage matrix is replaced by mineralized bone.Besides,from the resting chondrocytes to prehypertrophic chondrocytes both secrete collagen type 2(Col2),so the researchers often regard the collagen type 2 as a marker protein of chondrocytes.There are many factors in each zone of distal peripheral long bone growth plate to regulate the physiological activities,the most important factors include:PTHrP,Ihh and the signal pathway loop between PTHrP and Ihh(Fig.1c)As an very important signal pathway to regulate chondrocytes,PTHrTP/Ihh signal pathway loop has quite interesting charateristics in 4 zones of growth plate.The content of PTHrP from resting chondrocytes to terminal chondrocytes were decresing,while the Ihh is just the opposite.The content of Ihh from the resting chondrocytes to the terminal hypertrophic chondrocytes were increasing(Supplementary Fig.1c).The reasons of the phenomenon might be the two kinds of proteins regulate the different physiological functions.PTHrP mainly inhibit cell differentiation,remaining the cells to be rest,so in the resting chondrocytes are the highest,and Ihh is the promotion of cell differentiation,inhibition of cell division.So Ihh is highly expressed in prehypertrophic.In addition,the factors in different layers of chondrocytes have their own characteristics:PTHrP is highly expressed in the most distal cells of resting area,while Ptchl is little expressed in the middle of resting chondrocytes.But Ptchl is highly expressed in proliferative chondrocytes.What’s more,prehypertrophic chondrocytes highly expressed Ihh,and temminal hypertrophic chondrocytes highly expressed CO1X(Supplementary Fig.1b)Thus it is not difficult to see that PTHrP is often inhibit the expression of Ptchl and Ptchl is an important regulatory factor of cell proliferative zone.In addition,Ihh is an important regulatory factor in cartilage differentiation,while Co1X collagen is a key protein to evaluate matured chondrocytes.In addition,PPR as the PTHrP receptor was mainly expressed in the prehypertrophic chondrocytes.And also prehypertrophic chondrocytes highly expressed Ihh and little expressed PTHrP.Resting chondrocytes secret PTHrP will inhibit the proliferative chondrocytes transform to hypertrophic chondrocytes.This process is mostly mediated by PPR.As a result,it is not difficult to see that the PPR(Receptor of PTHrP)play an important role in inhibiting proliferative chondrocytes differentiation.mTOR pathway mainly regulate the important metabolism activity such as cell proliferation,differentiation,apoptosis and so on.It has been found two subtypes:mTORCl and mTORC2.The role of mTOR signal pathway on cartilage metabolism diseases has been confirmed.The first,mTOR can regulate the proliferation an apoptosis of chondrocytes,thus have an impact on chondrosaroma,arthritis disease and achondroplasia.Besides,mTOR can regulate the expression of inflammatory factors result in having an effect on autoimmune arthritis diseases.People have a profound understanding on the mTORCl signaling pathway result from the discovery of selective inhibitor of mTORCl(Rapamycin,RAPA).Rheb is a component of mTORC1,was originally found in nerve cells,and then has been found in other cells in sucession.Rheb includes two subtypes:Rhebl and Rheb2.The main function of Rheb is involved in Tsc1/2 component(Supplementary Fig.2)to participate regulating the function of mTORC1 signaling pathway.Rheb plays a positive regulator while Tsc1/2 component down regulates mTORC1 signaling pathway.As a result,to some degree Rheb can participate in the physiological activity such as prolifertatio,differentiation,apoptosis and immflatory of cells by mTORCl signaling pathway.P-S6 as a downstream protein of mTORC1 signal pathway,and is a very important signaling molecule for the function of mTORC1.It’s mainly involved in the synthesis of protein,finally promoting the different kinds of metabolism activities in cells.In practical study process,people measure the activity of mTORC1 by detecting the P-S6 quantity.As a result,it is not difficult to see that P-S6 is a very important protein factor for mTORC1.Objective:To investigate the function and importance of Rheb in the chondrocytes from the level of gene,we chose the persuasive,specificity and sensitivity Cre/LoxP gene targeting technology to explore this issue.This research has a good understanding of 3 days,7 days,10 days,14 days and 21 days mice gross appearances,the microstructure of distal long bone,growth plate of distal femur and the pathological of the chondrocytes when knocking out Rheb by the means of conditionally knocking out the Rheb gene in chondrocytes from the whole life,using high resolution CT(μ.-CT)of scanning,immunohistochemistry(IHC)staining,histological examination(H&E assay),safranin O-fast green staining,western blot(WB)and other technical means.This research will offer a new direction to diagnosis the cartilage development related diseases and find a new targeting of the medicine for curing the ACH.Methods:1.Generating mice with an chondrocytes-specific knockout of RhebWe purchased Co12α-cre transgenic mice from Jackson Laboratory(Jackson lab,America).The professor Xiao Bo from neural development and metabolism teaching and research office of Sichuan University Huaxi Medical College gave us the Rhebloxp/loxp(Rheb-/-)transgenic mice as a gift.Selecting the 8 weeks old male Rheb-/－ C57/BL6 background mice were crossed with 8 weeks old male Col2a-cre C57/BL6 background mice.Thus we can get the first generation which can be called hybridization mice(F1).We selected 8 weeks old Rheb+/--cre mice from the F1 generation mice to backcross,thus we can get the second generation(F2).Using the genotype idenfication to pick up the Rheb-/-cre knockout mice from F2,marked as the KO group,while the littermates such as Rheb-/-,Col2α-cre and Rheb+/--cre are the control mice,marked as the WT group.Thus continually using the F2 backcrossed can we get the Rheb-/--cre knockout mice.We used the genotping technology to pick out the targeted mice.The mice bred are in accord with the Mendel’s laws of hybridization.In addition,to understand the expression of Rheb protein and its downstream P-S6 protein,we using the WB technology to detect the costicartilage from the 24 hours new born mice both in KO and WT group so that we can see if the Rheb gene truly be knock out from the level of protein.2.Effect of Rheb gene in mouse cartilage developmentWe used 3 days old,7 days old,10 days old,14 days old and 21 days old knockout mice(KO)and littermates as the wild-type mice(WT)from the same feeding environment.Each group contained 3 mice,and 30 mice in total.We put the mice to death by intraperitoneal injecting the pentobarbital sodium.First of all,photographing the mice,weighting the body,visceras and tissues,measuring the body length,tail length,femur length and nose length,using the knee joints to do the expriments such as H&E,safranin O and fast green staining,IHC and WB detection on the 3 days old mouse costicartilage.In addition,we used the biostatistic to detect the different layers chondrocytes number of distal femur in H&E staining,safranin O and fast green staining show signs of dyeing area size(Aera),IHC reavealing the Mean optical density(MOD)of each protein expression intensity,WB measuring Gray value of each protein(Gray value,GV)to calculate the change of protein expression(a Fold change).The quantify results are measured by Image-Pro Plus(IPP,America).We detected the proteins quantification of distal long bone growth plate chondrocytes to see the impacts of body weight increasing and body length growing in the condition of knockout Rheb gene.3.The study about the role of Rheb gene in bone metabolism of miceTo understand the effect of mice distal femur’ s micro-structure,we used the 21 days old mice femur from KO group and WT group doing the Micro-CT scanning and three-dimensional reconstruction.To understand the effect of mice distal femur’s osteoclasts,we used the 21 days old mice femur from KO group and WT group doing the Tartrate-resistant acid phosphatase staining.Setting the parameters for the scanning voltage 70KV,power 30 W,scanning current was 429 μ.A,each scan thickness of 20 μm.Bone metabolism parameters after scanning analysis include the following:the trabecular thickness(Tb.Th);bone mineral density(BMD)and trabecular number(Tb.N).All these parameters were used to make sure the chondroctytes-specific knockout Rheb whether have an effect on the osteoblasts and osteoclasts in the distal femur.Results:1.Generating chondrocytes-specific knockout Rheb gene miceWe do the genotyping gene in the 3 days old mice,and found the Rheb gene is located on chromosome containing loxp fragments from the samples in KO mice,the production of PCR is 850 bp;while the production of PCR in one loxp floxed mice is 800 bp and 850 bp;in addition,the production of PCR was 800 bp in none loxp floxed mice.Besides,the production of PCR containing the Col2-cre is in 500 bp,suggesting that it is a Rheb-/－-cre mice which is totally knock out the Rheb gene.Thus we can make sure the Rheb gene is knocked out specific in chondrocytes.In addition,we used western blotting to detect the expression of Rheb protein from the costicartilage of the both KO and WT 24 hours old mice,and verify the effect of knockout from the level of protein.Besides,we detected the expression of Rheb downstream protein P-S6 at the same time,and it appeared the biggest difference.Compared with the WT group mice,the expression of P-S6 decreased obviously in KO group mice.From the other point,suggesting that the expression of P-S6 is inhibited obviously when Rheb gene knocked out.Rheb protein participates in the synthesis of P-S6 in the normal circumstances.The decreased expression of Rheb will inhibit the expression of P-S6.As a result we verify the effect of knockout mice from both the gene and protein level.It offered us a good basis to a futher study.In addition,we found that in 3～21 days old mice,compared KO mice with WT mice,the weight of body,adipose tissue,heart,liver,spleen,lung,kidney,brain and skeletal tissue of KO mice all decreased significantly,but the ratio of adipose tissue,heart,liver,spleen,lung,kidney and brain weight between body weight were not obviously different in KO group and WT group.While the ratio of skeletal tissue between body-weight in KO group is much lower than the WT group.All the information points out that the decreased obviously parts of body weight are the skeletal group.As a result,knocking out of Rheb gene will lead to the restriction of skeletal development..In addition,we found that compared with WT 3～21 days old mice,the body length and tail length of KO mice is shorter through the measurement of different group mice.And also we found that KO mice’s bridge of nose length were shorter than WT mice during the time of 7～21 days.At last,we found the femur length in KO mice were shorter than WT mice,especially in 21 days old.It is just because most of the cartilage tissue distributed around the vertebral epiphyseal plate,the growth plates of long bone,and nose bone.So it is not difficult to see that chondrocytes-specific knockout Rheb gene in mice have an inhibitory impact on the endochondral ossification.2、The research on the effect of Rheb gene in cartilage develomentDuring the 3～21 days old mice,Compared with the WT group mice,the KO group mice’s chondrocytes of growth plates arranged disorderly,and the boundery between different chondrocytes zone is not clear from the H&E staining of distal femur.According to the quantify of chondrocytes in growth plate from the H&E staining,the quantity of every chondrocytes zone decreased significantly,especially in the columnar chondrocytes,suggestion that the chondrocytes-specific knockout Rheb inhibited the cartilage development of growth plate the most.Proliferative chondrocytes are responsible for the precursor of chondrocytes to proliferate and divide.It plays an important role during the long bone development in mammal.If the body is in the puberty,the proliferative chondrocytes will continually proliferate to provide the chondrocytes for the growing of long bone and endochondral ossification.The opposite,if the proliferative chondrocytes’ development is delay or inhibited obviously,this might lead to short-limbs dwarfism.It is the same results from safranin O and fast green staining of the 3～21 days old mice’s femur.which proving our finding from another angle.First of all,the number of distal femur growth plate decreased obviously,and the cartilage’s develpment was inhibited especially in proliferative chondrocytes.Second,detecting the area of safranin O staining we can speculate the quantity of growth plate chondrocytes.Compared with WT group mice,the quantity of growth plate chondrocytes decreased too in KO group mice.To investigate the causation of KO mice proliferative zone chondrocytes decreased obviously and the quantity of chondrocytes decreased,we performed IHC experiments around distal femoral growth plate as well as peformed WB experiments using the sample from the protein of newborn mice costicartilage.The results of IHC experiments in 21 days old mice knee around growth plates is that parathyroid hormone-related peptide receptors(PTHrP receptor,PPR)expression level is significantly reduced.While the PPR is parathyroid gland hormone-related protein(Parathyroid Hormone Related Protein,PTHrP)receptor,mainly expressed in the prehypertrophic chondrocytes.Besides,the expression of PPR will directly contribute to the physiological function of the proliferative chondrocytes which means promote proliferation of proliferative chondrocytes.In addition,we found the expression of Indian hedgehog(Indian hedgehog protein,Ihh)and collagen type 10(Col X)decreased by using the western blotting experiment.It enlighten us the decreased of Ihh and Col X resulted from knockout of Rheb in KO group chondrocytes.3.The research on the role of Rheb gene in the bone metabolism from miceFirst,using H&E staining in mice 3～21 days,we found that compared with WT group,the bone trabeculas nearby mice growth plate are more sparse,and bone trabeculas and cortexes of bone are more thiner.It revealed us that the KO group mice had the osteoporosis.The reason might be that knocking out of Rheb gene lead to the sythesis of chondrocytes and function of secreting protein decreased,slowed down the metabolism and proliferrate in porliferative chondrocytes.So the ability of endochondral ossification was weak and finally caused the oestoporosis of distal femur.In addition,we performed p,CT in 21 days old mice distal femur,and found that compared with WT group mice,the Tb.N,and BMD decreased obviously,while the Tb.Sp inceased significantly.These results make us pretty sure that the KO group mice are osteoporsis.Besides we did the TRAP staining on the distal femur of 21 days old mice.We found that the osteoclasts of the bone trabecula around the growth plate decreased obviously,so we can see that knocking out Rheb gene lead to inhibit the function of oestoclast.In a word,it is not difficult to see that chondrocytes-specific knock out Rheb does not inhibit the function of osteoblast,but also inhibit the function of osteoclast.Conclusion:Chondrocytes-specific knock out Rheb gene in mice will lead to short-limbs dwarfism,the manifestation are ACH,accompanied by osteoporosis.The reasons are as follow:lacking of Rheb gene in chondrocytes lead to the level of P-S6 decreased,thus ribosomal phosphorylation proteins which can conduct the sythesis of proteins decrease,the related metabolism activity slowed.Besides,the expression of PPR,Ihh and ColX decreased will lead to slow down the proliferation in proliferative chondrocytes.And also the mature and differentiation process of cartilage will be inhibited.Finally,the cartilage development slowed down and the endochondral ossification decreased result from the preliferative zone offer just scarcity chondrocytes to prehypertrophic and hypertrophic chondrocytes,and the differentiation ability becomes week.Consequently,long bone development slow down and manifestation is short-limbs dwarfism and distal femur ostoporosis.In this study,the significance for this cause was providing the strong experimental evidence for ACH patient’s diagnosis from the respect of basis research,as well as a possible consolidation drug target for ACH and ACH with osteoporosis patients. |
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