Activated Effects of Parathyroid Hormone-Related Protein on Human Hepatic Stellate Cells
et al. (2013) Activated Effects of Parathyroid Hormone-Related Protein on Human Hepatic Stellate Cells. PLoS
ONE 8(10): e76517. doi:10.1371/journal.pone.0076517
Activated Effects of Parathyroid Hormone-Related Protein on Human Hepatic Stellate Cells
Fen-Fen Liang 0
Cui-Ping Liu 0
Li-Xuan Li 0
Min-Min Xue 0
Fang Xie 0
Yu Guo 0
Lan Bai 0
Rifaat Safadi, Haassah Medical Center, Israel
0 1 Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University , Guangzhou, Guangdong Province , China , 2 Department of Huiqiao Building, Nanfang Hospital, Southern Medical University , Guangzhou, Guangdong Province , China
Background & Aims: After years of experiments and clinical studies, parathyroid hormone-related protein(PTHrP) has been shown to be a bone formation promoter that elicits rapid effects with limited adverse reaction. Recently, PTHrP was reported to promote fibrosis in rat kidney in conjunction with transforming growth factor-beta1 (TGF-b1), which is also a fibrosis promoter in liver. However, the effect of PTHrP in liver has not been determined. In this study, the promoting actions of PTHrP were first investigated in human normal hepatic stellate cells (HSC) and LX-2 cell lines. Methods: TGF-b1, alpha-smooth muscle actin (a-SMA), matrix metalloproteinase 2 (MMP-2), and collagen I mRNA were quantified by real-time polymerase chain reaction (PCR) after HSCs or LX-2 cells were treated with PTHrP(1-36) or TGF-b1. Protein levels were also assessed by western-blot analysis. Alpha-SMA were also detected by immunofluorescence, and TGFb1 secretion was measured with enzyme-linked immunosorbent assay (ELISA) of HSC cell culture media. Results: In cultured human HSCs, mRNA and protein levels of a-SMA, collagen I, MMP-2, and TGF-b1 were increased by PTHrP treatment. A similar increasing pattern was also observed in LX-2 cells. Moreover, PTHrP significantly increased TGFb1 secretion in cultured media from HSCs. Conclusions: PTHrP activated HSCs and promoted the fibrosis process in LX-2 cells. These procedures were probably mediated via TGF-b1, highlighting the potential effects of PTHrP in the liver.
Funding: This work was supported by grants from the National Natural Scientific Foundation of China (#81170354) http://www.nsfc.gov.cn/. The funders had no
role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Parathyroid hormone-related protein (PTHrP) was first
identified from cancers that caused hypercalcemia, but over 25 years of
study, it has been demonstrated to work as a multifunctional
cytokine . However, studies of PTHrP have mainly focused on
bones and tumors. Experiments have demonstrated that PTHrP
promotes bone formation and is an excellent osteogenic promoter
that exerts rapid effects and few adverse reactions, even after years
of clinical treatments for osteoporosis (OP) [2,3,4,5]. Recently,
PTHrP was reported to promote renal fibrogenesis, with the
cooperation of TGF-b1 (transforming growth factor-b1), EGF
(endothelial growth factor), and VEGF (vascular endothelial
growth factor) . TGF-b1 is a powerful fibrosis promoter and
plays a central role in many fibrosis processes, including liver
During chronic liver disease, hepatic stellate cells (HSCs) is a
principal fibrogenic cell type that contributes to collagen
accumulation . Activation of HSCs is a key event in hepatic
fibrosis, where they acquire contractility and the extracellular
matrix (ECM) changes as they transform to myofibroblast-like cells
. These cells express the myofibroblast marker a-smooth muscle
actin (a-SMA) [9,10], and synthesize fibrillar collagens. The
initiation and persistence of HSC activation is regulated by many
signaling molecules, including TGF-b1 . HSC activation can
strongly produce TGF-b1 to maintain its elevated level, and
TGFb1 then activates and recruits more myofibroblasts to the injured
liver . This resulting in enhanced deposition of collagens into
the interstitial spaces, which finally impairs liver function .
PTHrP it is normally produced in every body tissue and organ,
including the liver [14,15]. Previous studies demonstrated that
PTHrP was markedly induced in hepatocytes during endotoxemia
and caused hepatic acute phase response [16,17]. These results
suggest that PTHrP may be an additional cytokine involved in
liver disease, but the exact effects of PTHrP on liver tissue is poorly
understood. Some chronic liver disease patients experience
endotoxemia. The hypothesis of the importance of endotoxins in
liver damage was first published in 1975 , and the critical role
of endotoxin in acute and chronic liver disease is now well
accepted and correlated with the disease severity . However,
the exactly effects of PTHrP in normal liver or in endotoxemia
have not yet been evaluated.
Datas regarding the effects of PTHrP on the liver or hepatic
diseases are rare. The aim of the present study was to obtain a
basic understanding of the effects of PTHrP in normal HSCs and
the activated LX-2 cell line. We show here for the first time that
PTHrP activates HSCs and promotes the fibrosis process of LX-2
cells, suggesting a role of the TGF-b1 system in promoting
Materials and Methods
HSC and LX-2 cell lines were obtained from the cell bank of
Sun Yat-sen University, Guangzhou, China. All these cells were
growth in Dulbeccos modified eagle medium (DMEM, from
Gibco,USA) with 10% FCS(fetal calf serum, from Gibco) in 5%
CO2 at 37uC. For all of the experiments, subconfluent cells (80%)
were incubated in 6-well dishes with either various PTHrP (136)
(Bachem, Bubendorf, Switzerland, H-3208) concentrations (0.1, 1,
10, 100 nM), or TGF-b1 (ProSpec, USA, CYT-716) of 1 ng/ml as
positive control, or 100 nM PTHrP with 1 ng/ml TGF-b1 in the
presence in DMEM with 2% FCS for different time periods (6, 12,
24, 48 h).
Western blot analysis
Cells were harvested in 0.2 ml of RIPA lysis buffer (Beyotime
Biotech, Nantong, China) with protease inhibitors (Roche,
Switzerland) and centrifuged with 12000 rpm for 20 min. The
supernatants were assayed for protein concentration (Beyotime
Biotech, Nantong, China). Protein samples were heated at 100uC
for 5 min before loading and 30 mg of the samples were subjected
to 10% sodium dodecyl sulphate-polyacrylamide gel
electrophoresis (SDS-PAGE) and then transferred to poly-vinylidene fluoride
(PVDF) membranes. Next, membranes were blocked with 5%
skimmed milk powder in TBST buffer (20 mM Tris, 500 mM
NaCl, and 0.1% Tween-20) for 1 h at room temperature with
gentle shaking. Membranes were then incubated overnight at 4uC
with various primary antibodies. The following primary antibodies
were used: 1:1000 mouse polyclonal anti-TGF-b1 (Abcam,
Cambridge, UK, ab64715), 1:1000 rabbit polyclonal anti
MMP2 (Sigma-Aldrich, St. Louis, MO, SAB4501891), 1:1000 rabbit
polyclonal anti-collagen I (Abcam, ab34710), 1:1000 rabbit
polyclonal anti-a-SMA (Santa Cruz Biotechnology, Santa Cruz,
CA, sc-130619).The membranes were washed with TBST buffer
and incubated in the appropriate peroxidase-conjugated
secondary antibody solution at a 1:5000 dilution (Zhongshan Biotech,
Beijing, China) before they were finally developed with enhanced
chemiluminescence (Millipore Corporation, USA, WBKLS0100).
The density of the individual bands was then quantified using a
densitometric scanner with Gel-pro Analyzer (Media Cybernetics,
mRNA expression analysis
Total RNA was isolated from HSCs and LX-2 cells with TRIzol
(TaKaRa Bio, Japan). cDNA was synthesized using the Revert Aid
First Stand cDNA Synthesis Kit(Fermentas, EU, #K1622) using
2 mg total RNA primed with random hexamer primers, following
the manufacturers instructions. The single-stranded cDNA was
amplified by comparative quantitative real-time RT-polymerase
chain reaction (PCR) using SYBR green Master Mix kit (Roche,
USA, Cat. No. 04887352001) on an Roche LightCycler 480.
Primers were as follows: TGF-b1, (Forward) 59- ACC TGA ACC
CGT GTT GCT CT -39 and (Reverse) 59- CTA AGG CGA AAG
CCC TCA AT -39; MMP-2, (Forward) 59-GTA TTT GAT GGC
ATC GCT CA -39 and (Reverse) 59- CAT TCC CTG CAA AGA
ACA CA -39; collagen I, (Forward) 59-GAA CGC GTG TCA
TCC CTT GT -39 and (Reverse) 59- GAA CGA GGT AGT CTT
TCA GCA ACA -39; a-SMA, (Forward) 59-TCG CAT CAA
GGC CCA AGA AA -39 and (Reverse) 59-GCT TCA CAG GAT
TCC CGT CTTA -39; GAPDH, (Forward) 59- TGC ACC ACC
AAC TGC TTA GC -39 and (Reverse) 59- GGC ATG GAC TGT
GGT CAT GAG -39. The cycles for PCR were as follows: one
cycle of 95uC for 10 minutes, 45 cycles of 15 seconds at 95uC, 1
minute at 62uC and a final 1 minute at 72uC. The mRNA
expression levels of the target genes were normalized to GAPDH.
Cells were plated on glass coverslips in 12-well culture dishes
and grown to approximately 50% confluence to promote cell
adherence of cells. After stimulation with PTHrP, the cells were
then washed twice with cold phosphate-buffered saline (PBS) and
then fixed in 4% paraformaldehyde in PBS for 10 minutes. After
fixation, cells were washed twice with PBS and then permeabilized
with PBS containing 0.1% Triton X-100 for 15 minutes. They
were then washed three times with PBS and incubated with
blocking solution (5% BSA in PBS) for 30 minutes. Primary
antibody a-SMA (Santa Cruz Biotechnology, sc-130619, diluted
1:200 in blocking solution) was incubated with the cells over night
at 4uC. After three washes with PBS, cells were incubated with
appropriate fluorescein-labeled IgG (Vector Laboratories, diluted
1:500 in blocking buffer) for 1 h. Cells were washed three times
with PBS and then stained with 5 mg/ml DAPI
(49,6-diamidino-2phenylindole, Beyotime Biotech, Nantong, China) for 2 minutes
and washed three times with PBS. Cells were viewed with a Nikon
Eclipse E600 fluorescence microscope.
Cell-conditioned medium protein assay
TGF-b1 protein was measured in the HSC-conditioned
medium after treatment with PTHrP(136) (10100 nM) for
Figure 2. Increased production of a-SMA in HSC and LX-2 cells after PTHrP(136) treatment. After treatment with 100 nM of PTHrP(136)
for 48 h, the a-SMA protein (43 kd) levels assessed by western blot (A) and mRNA levels measured by q-PCR were increased in HSCs (B). The same
patterns were observed in LX-2 (C and D). * p,0.05 compared to the control group.
2448 h, using a commercial enzyme-linked immunosorbent assay
(ELISA, eBioscience, San Diego, CA, E13702-107) following the
manufacturers instructions. Total TGF-b1 was determined in
100 ml of the cell-conditioned medium (stored at 280uC). Inactive
TGF-b1 was converted to the active form by incubating these cell
culture supernatants with 1 N HCl for 10 min, followed by
neutralization with 1 N NaOH. Protein content was determined
by the bicinchoninic acid (BCA) method (Pierce). TGF-b1
concentrations were quantified by comparison with a standard
curve of human TGF-b1.
Data are presented as mean 6 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)
13.0 software (Chicago, IL). Probability (p)-values less than 0.05
were considered statistically significant.
Increased production of a-SMA in HSCs and LX-2 cells
after treatment with PTHrP(136)
To explore the possible effects of PTHrP in HSCs, we
performed immunostaining for a-SMA. After incubation with
100 nM PTHrP(136). HSCs stained strongly for a-SMA, and
LX-2 staining significantly increased compared with control
(Figure 1). After treated with various concentrations (0.1
100 nM) PTHrP(136) for different time periods (648 h), levels
of a-SMA mRNA (by q-PCR) and protein (western-blot) were
found to be increased at 10100 nM for 2448 h in both types of
cells, with 1.3- to 3- fold increases (Figure 2). At 10100 nM
PTHrP for 612 h, both cell types showed 0.9- to 1.1-fold
increases, but there were no significant difference in these groups.
Similar results were obtained for the 0.11 nM PTHrP groups.
The strong expression of a-SMA, which is a myofibroblast marker,
showed that PTHrP activates HSCs. All experiments were
repeated at least three times.
Figure 3. Increased production of collagen I in HSC and LX-2 cells after treatment with PTHrP(136). After treatment with PTHrP(136)
(100 nM) for 48 h, collagen I (138 kd) protein levels were assessed by western-blot (A); mRNA expression was measured with q-PCR and found to be
increased in both HSCs (B), and LX-2 cells (C and D). * p,0.05 compared to the control group.
PTHrP(136) induced MMP-2 and collagen I mRNA and
protein production in HSCs and LX-2 cells
Collagen I is the main component of the ECM, and activated
HSCs are a major source of collagen type I. In response to
PTHrP(136) at 10100 nM for 2448 h, the collagen I protein
(138 kd) was significantly increased 2- to 2.5-fold in HSCs, and its
mRNA levels also increased by 2- to 2.5-fold as assessed by
realtime PCR compared to untreated control cells (Figure 3). In LX-2
cells, exposure to PTHrP (136) at 10100 nM for 48 h,
stimulated collagen I protein and mRNA levels increased by
1.8to 4-fold, while treatment with PTHrP for 24 h at 100 nM made
these increases statistically significant (Figure 3). The other
concentrations and time points failed to reach statistical
significance in both cell types compared to the control groups. The
comparison of TGF-b1 alone to TGF-b1 with PTHrP was not
statistically significant with regard to collagen I expression
MMP expression is increased in liver fibrosis, and MMP-2 is
expressed and secreted by activated HSCs. After PTHrP was
provided at 10100 nM for 2448 h, western-blot analysis of
whole cell lysates demonstrated that MMP-2 protein (72 kd) levels
were increased in both HSC and LX-2 cells by 1.5- to 3-fold.
Similarly, we observed increased expression of MMP-2 mRNA by
1.5- to 3-fold in both cell types as assessed by comparative real
time PCR (Figure 5). The other concentrations and time points did
not show significant differences with regard to MMP-2 levels in
either cell type. At least three independent experiments were used
for the statistical analyses.
PTHrP(1-36) induced TGF-b1 secretion in HSC and LX-2
It is well accepted that TGF-b1 is a cytokine that plays a central
role in fibrosis, and activated HSCs themselves can secrete
TGFb1. We evaluated whether PTHrP would affect TGF-b1
production. We found that TGF-b1 protein (45 kd) in HSCs
was stimulated by PTHrP(136) at 10100 nM concentrations as
early as 24 h, and 100 nM treatment of LX-2 cells at 48 h, with
both cell types exhibiting 2- to 3.5-fold changes. TGF-b1 mRNA
levels increased by 2- to 3-fold (Figure 6). TGF-b1 secretion was
also measured in HSC cultured medium by ELISA. As expected,
this factor was elicited by PTHrP(136) at concentrations of 10
100 nM for 2448 h by 2- to 3-fold (Figure 7). These data suggest
that TGF-b1 in HSCs is stimulated by PTHrP(136). At least
Figure 4. Collagen I expression in HSCs and LX-2 cells after TGF-b1 and PTHrP treatment. After treatment with TGF-b1 (1 ng/ml) alone or
with PTHrP (100 nM) for 48 h, collagen I (138 kd) total protein was assessed by western blot (A); mRNA expression was measured with q-PCR and
found to be increased in both HSCs (B), and LX-2 cells (C and D). * p,0.05 compared to the control group. No significant difference was observed
between treated groups.
PTHrP was initially identified in cancers that caused lethal
paraneoplastic humoral hypercalcemia . PTHrP has a similar
structure with parathyroid hormone (PTH), in term of N-terminal
amino acid sequence homology and that fact that its full biological
activity is contained within the first 34 amino acids . In the
previous organ-focused investigations, PTHrP was found to be
produced in almost every tissue and organ in the body, including
heart, brain, skeletal muscle, bladder, lung, bile duct, immune
system, liver, uterus, and testes, as well as most endocrine organs
including the pituitary, thyroid gland C-cells, and gastric mucosa
enterochromaffin-like cells [14,15,22]. As early as 1996, it was
reported that PTHrP gene expression was induced in rat vital
organs, including liver, spleen, heart, lung, and kidney in response
to LPS (lipopolysaccharide) injection [17,23]. Hepatic PTHrP
mRNA levels were acutely induced in rat liver in response to a
near lethal dose of endotoxin (LPS), and its protein production was
also markedly induced in periportal hepatocytes . It was
already established that endotoxins are a critical cofactor in acute
and chronic liver disease in both experimental and clinical settings,
and their levels correlated with disease severity . These
findings suggest that PTHrP may be an additional cytokine
involved in liver disease. However, the exact effects of PTHrP in
liver cells had not been evaluated.
PTHrP was recently demonstrated to promote fibrogenesis in
the obstructed mouse kidney, and it seemed to act in conjunction
with TGF-b1, EGF, and VEGF [6,24]. In our study, we first
examined what effects PTHrP might exert in two commercial cell
lines. We detected that the activated marker a-SMA was
upregulated by PTHrP treated of HSCs, which was strongly
suggestive of activation. Collagen I is a composition of ECM,
and MMP-2 degrades the ECM. The previous two factors are
mainly produced by activated HSCs and further activate stellate
cell growth . In fact, we observed that both collagen I and
MMP-2 were increased in PTHrP treated cells compared with
control HSCs. Therefore, PTHrP seems to be related to activation
changes in HSCs.
LX-2 cells are an activated line that expresses increased levels of
a-SMA in contrast to normal stellate cells . However, we still
observed increased a-SMA immunoreactivity in PTHrP treated
cells by immunofluorescence. We also found increased induction
of collagen I, MMP-2, and TGF-b1 mRNA and protein levels in
Figure 5. Increased production of MMP-2 in HSCs and LX-2 cells after PTHrP(136) treatment. After a 48 h treatment with 100 nM of
PTHrP(136), the MMP-2 (72 kd) total protein levels were assessed by western blot (A); mRNA expression was measured with q-PCR and found to be
increased in HSC (B). Similar patterns were observed for LX-2 cells (C and D). * p,0.05 compared to the control group.
LX-2 cells, but the stimulated effects of collagen I required higher
PTHrP concentrations and longer time periods; 10 nM PTHrP
treatment for 24 h did not have statistically significant effects. Our
results suggest that PTHrP is likely to regulate fibrogenesis by
affecting LX-2 cells. However, an in vivo study of human plasma
concentrations of PTHrP did not shown an increase in the
presence of hepatic cirrhosis, even with severe cases . It is well
known that PTHrP can act in endocrine, intracrine, and
paracrine, but especially in the latter . Although plasma PTHrP
was not significantly increased in hepatic cirrhosis patients, we still
observed HSC and LX-2 activation in vitro. These results suggest
that PTHrP probably acts in a paracrine fashion. However,
elucidation of this cytokine in the intact liver will require further
TGF-b1 plays a central role in liver fibrosis, it can induce
differentiation of HSCs into collagen producing myofibroblasts. In
turn, activated HSCs themselves can secrete TGF-b1, which
increases hepatocytes damage . Our in vitro data strongly
support the hypothesis that PTHrP contributes to TGF-b1
overexpression in HSCs. It seems that PTHrP may have mediated its
effects through TGF-b1 signaling because PTHrP with TGF-b1
did not induce further up-regulation of collagen I compared to
TGF-b1 alone. Thus, the present findings also suggest that the
activated effects induced by PTHrP in this setting might be
mediated, at least in part, by the TGF-b1 system.
The first recognition of PTHrP was related to calcium
metabolism, but is was subsequently found to be a potent anabolic
agent and was considered as a potential OP treatment in humans
[27,28]. An important complication of chronic liver disease is
osteodystrophy, which includes OP, but the general treatment of
OP in chronic liver disease is not satisfactory . PTHrP
selectively and rapidly stimulates bone formation and has less
adverse effects than other options, making it a potential option in
treating OP. Moreover, its safety has been partially evaluated but
these tests were mostly focused on calcium metabolism [2,30].
Data regarding the effects on other organs and tissues are limited.
Contrary to the fibrogenesis effects of PTHrP in kidney, it has
been suggested that PTHrP increases renal plasma flow but does
not regulate systemic hemodynamics in healthy humans . It is
unclear what effects would be observed in liver, therefore, more
studies are needed.
In summary, we show here for the first time the activating
effects of PTHrP in HSCs, and its impact on activated LX-2 cells.
Our results suggest a role for the TGF-b1 system as a mediator of
Figure 6. PTHrP(136) induced TGF-b1 in HSC and LX-2 cells. After a 48 h treatment with 100 nM of PTHrP(136), TGF -b1 (45 kd) total
protein levels were assessed by western-blot (A); mRNA expression was measured with q-PCR and found to be increased in HSC (B), and LX-2 (C and
D). * p,0.05 compared to the control group.
these effects and support the notion that PTHrP may act as an
additional cytokine in liver disease. Further work will assess the
relative mechanisms to the actions of PTHrP in this setting.
Conceived and designed the experiments: LB FFL. Performed the
experiments: FFL CPL LXL MMX FX YG. Analyzed the data: LB
FFL. Contributed reagents/materials/analysis tools: FFL CPL LXL MMX
FX YG. Wrote the paper: FFL.
1. McCauley LK , Martin TJ ( 2012 ) Twenty-five years of PTHrP progress: From cancer hormone to multifunctional cytokine . J Bone Miner Res 27 : 1231 - 1239 .
2. Horwitz MJ , Tedesco MB , Garcia-Ocana A , Sereika SM , Prebehala L , et al. ( 2010 ) Parathyroid hormone-related protein for the treatment of postmenopausal osteoporosis: defining the maximal tolerable dose . J Clin Endocrinol Metab 95 : 1279 - 1287 .
3. Bisello A , Horwitz MJ , Stewart AF ( 2004 ) Parathyroid hormone-related protein: an essential physiological regulator of adult bone mass . Endocrinology 145 : 3551 - 3553 .
4. Horwitz MJ , Tedesco MB , Gundberg C , Garcia-Ocana A , Stewart AF ( 2003 ) Short-term, high-dose parathyroid hormone-related protein as a skeletal anabolic agent for the treatment of postmenopausal osteoporosis . J Clin Endocrinol Metab 88 : 569 - 575 .
5. Wysolmerski JJ ( 2012 ) Parathyroid hormone-related protein: an update . J Clin Endocrinol Metab 97 : 2947 - 2956 .
6. Ardura JA , Rayego-Mateos S , Ramila D , Ruiz-Ortega M , Esbrit P ( 2010 ) Parathyroid hormone-related protein promotes epithelial-mesenchymal transition . J Am Soc Nephrol 21 : 237 - 248 .
7. Friedman SL ( 2000 ) Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury . J Biol Chem 275 : 2247 - 2250 .
8. Xu L , Hui AY , Albanis E , Arthur MJ , O9Byrne SM , et al. ( 2005 ) Human hepatic stellate cell lines, LX-1 and LX-2: new tools for analysis of hepatic fibrosis . Gut 54 : 142 - 151 .
9. Bataller R , Brenner DA ( 2005 ) Liver fibrosis . J. Clin. Invest 115 : 209 - 218 .
10. Bataller R , Brenner DA ( 2001 ) Hepatic stellate cells as a target for the treatment of liver fibrosis . Semin Liver Dis 21 : 437 - 452 .
11. Gressner AM , Weiskirchen R , Breitkopf K , Dooley S ( 2002 ) Roles of TGF-beta in hepatic fibrosis . Front Biosci 7 : d793 - d807 .
12. Kisseleva T , Brenner DA ( 2007 ) Role of hepatic stellate cells in fibrogenesis and the reversal of fibrosis . J Gastroenterol Hepatol 22 Suppl 1 : S73 - S78 .
13. Friedman SL ( 2008 ) Hepatic fibrosis-Overview . Toxicology 254 : 120 - 129 .
14. Philbrick WM , Wysolmerski JJ , Galbraith S , Holt E , Orloff JJ , et al. ( 1996 ) Defining the roles of parathyroid hormone-related protein in normal physiology . Physiol Rev 76 : 127 - 173 .
15. Urena P , Kong XF , Abou-Samra AB , Juppner H , Kronenberg HM , et al. ( 1993 ) Parathyroid hormone (PTH)/PTH-related peptide receptor messenger ribonucleic acids are widely distributed in rat tissues . Endocrinology 133 : 617 - 623 .
16. Funk JL , Moser AH , Grunfeld C , Feingold KR ( 1997 ) Parathyroid hormonerelated protein is induced in the adult liver during endotoxemia and stimulates the hepatic acute phase response . Endocrinology 138 : 2665 - 2673 .
17. Funk JL , Krul EJ , Moser AH , Shigenaga JK , Strewler GJ , et al. ( 1993 ) Endotoxin increases parathyroid hormone-related protein mRNA levels in mouse spleen . Mediation by tumor necrosis factor . J Clin Invest 92 : 2546 - 2552 .
18. JP N ( 1975 ) The role of endotoxin in liver injury . Gastroenterology 69 : 1346 - 1356 .
19. Nolan JP ( 2010 ) The role of intestinal endotoxin in liver injury: a long and evolving history . Hepatology 52 : 1829 - 1835 .
20. Iredale JP ( 2001 ) Hepatic stellate cell behavior during resolution of liver injury . Semin Liver Dis 21 : 427 - 436 .
21. Kemp BE , Moseley JM , Rodda CP , Ebeling PR , Wettenhall RE , et al. ( 1987 ) Parathyroid hormone-related protein of malignancy: active synthetic fragments . Science 238 : 1568 - 1570 .
22. Liu C , Chen J , Guo Y , Yang L , Zhao C , et al. ( 2011 ) The expression of PTHLH in human gastric mucosa enterochromaffin-like cells . Dig Dis Sci 56 : 993 - 998 .
23. Funk JL , Moser AH , Strewler GJ , Feingold KR , Grunfeld C ( 1996 ) Parathyroid hormone-related protein is induced during lethal endotoxemia and contributes to endotoxin-induced mortality in rodents . Mol Med 2 : 204 - 210 .
24. Ardura JA , Berruguete R , Ramila D , Alvarez-Arroyo MV , Esbrit P ( 2008 ) Parathyroid hormone-related protein interacts with vascular endothelial growth factor to promote fibrogenesis in the obstructed mouse kidney . Am J Physiol Renal Physiol 295 : F415 - F425 .
25. Fabrega E , Rivero M , Pons-Romero F , Garcia-Unzueta MT , Amado JA ( 2000 ) Parathyroid hormone-related protein in liver cirrhosis . Dig Dis Sci 45 : 703 .
26. Breitkopf K , Haas S , Wiercinska E , Singer MV , Dooley S ( 2005 ) Anti-TGF-beta strategies for the treatment of chronic liver disease . Alcohol Clin Exp Res 29 : 121S - 131S .
27. Henry JG , Mitnick M , Dann PR , Stewart AF ( 1997 ) Parathyroid hormonerelated protein-(1-36) is biologically active when administered subcutaneously to humans . J Clin Endocrinol Metab 82 : 900 - 906 .
28. Horwitz MJ , Tedesco MB , Gundberg C , Garcia-Ocana A , Stewart AF ( 2003 ) Short-term, high-dose parathyroid hormone-related protein as a skeletal anabolic agent for the treatment of postmenopausal osteoporosis . J Clin Endocrinol Metab 88 : 569 - 575 .
29. Collier JD , Ninkovic M , Compston JE ( 2002 ) Guidelines on the management of osteoporosis associated with chronic liver disease . Gut 50 Suppl 1 : i1 - i9 .
30. Horwitz MJ , Tedesco MB , Sereika SM , Prebehala L , Gundberg CM , et al. ( 2011 ) A 7-day continuous infusion of PTH or PTHrP suppresses bone formation and uncouples bone turnover . J Bone Miner Res 26 : 2287 - 2297 .
31. Wolzt M , Schmetterer L , Dorner G , Zelger G , Entlicher J , et al. ( 1997 ) Hemodynamic effects of parathyroid hormone-related peptide-(1-34) in humans . J Clin Endocrinol Metab 82 : 2548 - 2551 .