in studying a relatively well-differentiated human hepatoma cell line (Hep G2),we found that the cells expressed mRNA for PTH-related peptide (PTHrP) and secreted biologically active PTHrP. In the present study, using...in studying a relatively well-differentiated human hepatoma cell line (Hep G2),we found that the cells expressed mRNA for PTH-related peptide (PTHrP) and secreted biologically active PTHrP. In the present study, using RT/PCR and Northern analysis,we found that the Hep G2 liver cells also express mRNA for the PTH/PTHrP receptor and exhibit specific binding for radiolabeled N-terminal PTHrP. Therefore, we hypothesized:the cells would respond to endogenously produced peptide. Since PTHrP has been implicated as a potential regulator of cell growth, we asked whether PTHrP might act in an autocrine/paracrine fashion to affect growth of Hep G2 cells. Endogenous PTHrP production by the cells in culture was neutralized by adding aliquots of polyclonal antiserum to either synthetic PTHrP(1-34)or recombinant PTHrP(-5 to 139) to the cultured cells. Both antisera were shown to be capable of inhibiting the ability of conditioned growth medium to stimulate cAMP accumulation in ROS cells in a manner similar to the inhibition produced by the PTHrP antagonist,[Asn10 Leu11,d-Trp12]PTHrP (7-34).When subconfluent Hep G2 cells were exposed to increasing amounts of these two rabbit antisera (1:800￣1:100 dilution in growth medium)for 3 days,a dose-dependent 40%￣ 50% increase in cell growth was observed (vs treatment with nonimmune rabbit serum).The increased cell growth produced by the antisera could be inhibited by concurrent addition of a large concentration (10 μmol/L)of synthetic PTHrP(1-36).The results show that addition of antisera which can neutralize the N-terminal biologic activity of PTHrP caused an enhanced growth of HeP G2 cells in culture which could be inhibited by addition of synthetic N-terminal PTHrP.The findings suggest a possible local regulatory role for PTHrP in liver growth.展开更多
Physiologic roles of PTHrP remain elusive,but some have implied a role of growth and differentiation.Since intestinal epithelial cell show orderly growth and differentiation as they proliferate in the crypt and migrat...Physiologic roles of PTHrP remain elusive,but some have implied a role of growth and differentiation.Since intestinal epithelial cell show orderly growth and differentiation as they proliferate in the crypt and migrate to the villus tip,we asked whether they might exhibit differences in expression of mRNA for either PTHrP or its receptor.AT/PCR was used to generate cDNA probe for either PTHrP or the PTH/PTHrP receptor.Total RNA was prepared from epithelial cells isolated form various region of rat gut and epithelial cell lines.derived from rat crypt(IEC-6)and human colon(LoVo)as wellas cell fractions taken sequentially along the villus-crypt axis of rat jejunum.The 1.6kb mRNA for PTHrP was detected in epithelia from all regions of rat gut(duodenum,jejunum,ileum,colon),in all fractions along the iejnnal villus tipcrypt axis,and in both cell lines.Likewise mRNA for the PTH/PTHrP receptor also was expressed,lbeit at lower level,in all regions,along the villus,and in both cell lines. Interestingly,while in kidney(positive control)two transcripts(1.5 & 2.4 kb)were detected as other reported,in intestinal epithelia and cell lines,only 1.skb transcript was evident.We conclude that mRNAs for both PTHrP and PTH/PTHrP receptor are expressed throughout the gut and that no obvious pattern of expre.ssion emerges from examining epithelia or cell lines representing different stage of differentiation. The role of PTHrP in gut epithelia remains to be defined.展开更多
In studying a relatively well-differentiated human hepatoma cell line (Hep G2),we found that the cells produced and secreted biologically active PTHrP.The present study was designed to determine whether PTHrP producti...In studying a relatively well-differentiated human hepatoma cell line (Hep G2),we found that the cells produced and secreted biologically active PTHrP.The present study was designed to determine whether PTHrP production by Hep G2 cells could be altered by agents that affect cell growth.PTHrP production was assessed by measuring immunoreactive peptide in culture medium using the Nichols immunoradiometric assay and by evaluating PTHrP mRNA levels in cells using Northern analysis. Treatment with 10μmol/L hydrocortisone or 10μg/L TGF-βfor 3 days inhibited Hep G2 cell growth by (28±6)%and(36)2) fi respectively and increased PTHrP in medium by (128 ±10)% and (525 ±27)% respectively.Related studies showed that the increase in PTHLP produced by both agents was dose-and time-dependent and that the increase in peptide in the medium was accompanied by an increase in PTHrP mRNA in the cells which also was dose- and timedependent. In contrast,culture of HeP G2 cells for 3 days in 10% fetal boxrine serum(FBS)or in high glucose(4 g/L)significantly increased cell growth by(38±6)%(vs no serum)and by(43±1)%(vs 1 g/L glucose) and suppressed PTHrP in the culture medium by (49±4)% and(55±0.4)%, respectively. The inhibition of PTHrP was found to be dose-and time-dependent,but FBS only marginally suppressed PTHrP mRNA levels and glucose did not detectably alter PTHrP mRNA. The results show that PTHrP production and secretion in HeP G2 cells can be regulated by factors that affect growth of the cells in culture. Agents which suppressed cell growth enhanced PTHrP production, while those that stimulated cell growth were associated with reduced PTHrP in medium. The findings imply that PTHrP may be involved in the altered cell growth produced by these factors; if so,the peptide appears to act as a suppressor of Hep G2 cell growth.展开更多
文摘in studying a relatively well-differentiated human hepatoma cell line (Hep G2),we found that the cells expressed mRNA for PTH-related peptide (PTHrP) and secreted biologically active PTHrP. In the present study, using RT/PCR and Northern analysis,we found that the Hep G2 liver cells also express mRNA for the PTH/PTHrP receptor and exhibit specific binding for radiolabeled N-terminal PTHrP. Therefore, we hypothesized:the cells would respond to endogenously produced peptide. Since PTHrP has been implicated as a potential regulator of cell growth, we asked whether PTHrP might act in an autocrine/paracrine fashion to affect growth of Hep G2 cells. Endogenous PTHrP production by the cells in culture was neutralized by adding aliquots of polyclonal antiserum to either synthetic PTHrP(1-34)or recombinant PTHrP(-5 to 139) to the cultured cells. Both antisera were shown to be capable of inhibiting the ability of conditioned growth medium to stimulate cAMP accumulation in ROS cells in a manner similar to the inhibition produced by the PTHrP antagonist,[Asn10 Leu11,d-Trp12]PTHrP (7-34).When subconfluent Hep G2 cells were exposed to increasing amounts of these two rabbit antisera (1:800￣1:100 dilution in growth medium)for 3 days,a dose-dependent 40%￣ 50% increase in cell growth was observed (vs treatment with nonimmune rabbit serum).The increased cell growth produced by the antisera could be inhibited by concurrent addition of a large concentration (10 μmol/L)of synthetic PTHrP(1-36).The results show that addition of antisera which can neutralize the N-terminal biologic activity of PTHrP caused an enhanced growth of HeP G2 cells in culture which could be inhibited by addition of synthetic N-terminal PTHrP.The findings suggest a possible local regulatory role for PTHrP in liver growth.
文摘Physiologic roles of PTHrP remain elusive,but some have implied a role of growth and differentiation.Since intestinal epithelial cell show orderly growth and differentiation as they proliferate in the crypt and migrate to the villus tip,we asked whether they might exhibit differences in expression of mRNA for either PTHrP or its receptor.AT/PCR was used to generate cDNA probe for either PTHrP or the PTH/PTHrP receptor.Total RNA was prepared from epithelial cells isolated form various region of rat gut and epithelial cell lines.derived from rat crypt(IEC-6)and human colon(LoVo)as wellas cell fractions taken sequentially along the villus-crypt axis of rat jejunum.The 1.6kb mRNA for PTHrP was detected in epithelia from all regions of rat gut(duodenum,jejunum,ileum,colon),in all fractions along the iejnnal villus tipcrypt axis,and in both cell lines.Likewise mRNA for the PTH/PTHrP receptor also was expressed,lbeit at lower level,in all regions,along the villus,and in both cell lines. Interestingly,while in kidney(positive control)two transcripts(1.5 & 2.4 kb)were detected as other reported,in intestinal epithelia and cell lines,only 1.skb transcript was evident.We conclude that mRNAs for both PTHrP and PTH/PTHrP receptor are expressed throughout the gut and that no obvious pattern of expre.ssion emerges from examining epithelia or cell lines representing different stage of differentiation. The role of PTHrP in gut epithelia remains to be defined.
文摘In studying a relatively well-differentiated human hepatoma cell line (Hep G2),we found that the cells produced and secreted biologically active PTHrP.The present study was designed to determine whether PTHrP production by Hep G2 cells could be altered by agents that affect cell growth.PTHrP production was assessed by measuring immunoreactive peptide in culture medium using the Nichols immunoradiometric assay and by evaluating PTHrP mRNA levels in cells using Northern analysis. Treatment with 10μmol/L hydrocortisone or 10μg/L TGF-βfor 3 days inhibited Hep G2 cell growth by (28±6)%and(36)2) fi respectively and increased PTHrP in medium by (128 ±10)% and (525 ±27)% respectively.Related studies showed that the increase in PTHLP produced by both agents was dose-and time-dependent and that the increase in peptide in the medium was accompanied by an increase in PTHrP mRNA in the cells which also was dose- and timedependent. In contrast,culture of HeP G2 cells for 3 days in 10% fetal boxrine serum(FBS)or in high glucose(4 g/L)significantly increased cell growth by(38±6)%(vs no serum)and by(43±1)%(vs 1 g/L glucose) and suppressed PTHrP in the culture medium by (49±4)% and(55±0.4)%, respectively. The inhibition of PTHrP was found to be dose-and time-dependent,but FBS only marginally suppressed PTHrP mRNA levels and glucose did not detectably alter PTHrP mRNA. The results show that PTHrP production and secretion in HeP G2 cells can be regulated by factors that affect growth of the cells in culture. Agents which suppressed cell growth enhanced PTHrP production, while those that stimulated cell growth were associated with reduced PTHrP in medium. The findings imply that PTHrP may be involved in the altered cell growth produced by these factors; if so,the peptide appears to act as a suppressor of Hep G2 cell growth.
