Editorials
Retinoids May Increase Fibrotic Potential of TGF-b: Crosstalk
Between Two Multi-functional Effectors
SEE ARTICLE ON PAGE 913
Key events in liver fibrosis include the accumulation of
extracellular matrix, the activation of hepatic stellate cells
(HSC) to proliferating myofibroblast-like cells, and a reduction in stellate-cell lipid droplets containing retinyl esters
(vitamin A).1-3 These findings in vivo are recapitulated in
culture, in which rat stellate cells grown on uncoated plastic
lose retinyl esters and transform into proliferating myofibroblast-like cells with a 10-fold increased secretion of collagen (mainly type I). The cytokine, transforming growth factor b (TGF-b) has a central pro-fibrogenic role; it may act
in a predominantly autocrine manner such that its release
specifically by stellate cells is central to fibrogenesis.1-3
The role of retinoids (normal analogs, synthetic analogs,
and metabolites of retinol) in liver fibrosis has become intriguing. In vitro, the activation of quiescent stellate cells
to dividing myofibroblast-like cells may be inhibited by the
addition of retinol and retinoic acid to the medium; in that
process, proliferation and collagen production is effectively
inhibited and, if retinol is administered, large fat-droplets
and long cellular processes are induced.4-5 Retinoic acid is
generally 100 to 1,000 times more potent than is retinol in
its anti-proliferative potential, suggesting that the effect of
retinol is mediated by the conversion of retinol to retinoic
acid.4-6
Parallel data in vivo have been reported by Senoo and
Wake;7 in their study, they injected 15 mg retinol/kg of body
weight into rats subcutaneously twice a week for 2 weeks
before repeated injection of carbon tetrachloride (CCl4 ). The
treatment resulted in increased storage of retinyl esters in
HSC and suppressed experimental hepatic fibrosis induced
by CCl4 . Beta-carotene treatment, which also increases retinyl ester storage in stellate cells, similarly attenuates CCl4 induced hepatic fibrosis.8 Consistent with this, vitamin A
deficiency, with low levels of retinyl esters in stellate cells,
promotes CCl4 -induced liver fibrosis.9
In this issue of HEPATOLOGY, Okuno et al.10 report in a
well-designed, technically sound study that retinoids exacerbate liver fibrosis, an observation that appears in conflict
with the observations discussed above. First, day-7 cultures
of stellate cells, in which the majority of cells have a myofibroblast-like phenotype, increased their secretion of total
Abbreviations: TGF-b, transforming growth factor b; PA, plasminogen activator;
CCl4 , carbon tetrachloride; RAR, retinoic acid receptor; RXR, retinoid X receptor;
CRBP, cellular retinol-binding protein.
From the Institute for Nutrition Research, School of Medicine, University of Oslo,
Oslo, Norway.
Received July 15, 1997; accepted August 7, 1997.
Address reprint requests to: Rune Blomhoff, Ph.D., Institute for Nutrition Research,
School of Medicine, University of Oslo, P.O. Box 1046, Blindern, 0316 Oslo, Norway.
Fax: 47-2285-1396.
Copyright q 1997 by the American Association for the Study of Liver Diseases.
0270-9139/97/2604-0040$3.00/0
TGF-b upon treatment with 9-cis or all-trans retinoic acid.
The effect of retinoic acid could be observed in as little as 3
hours after its addition to the cells. Second, treated cells
released active TGF-b into the culture medium. These data
demonstrate that retinoic acid both induces the secretion of
latent TGF-b and stimulates its activation. Finally, the authors found that surface-associated plasminogen activator
was increased in cultures exposed to 9-cis retinoic acid, suggesting that plasmin may mediate the activation of latent
TGF-b in this system.
A review of retinoid metabolism and signaling is necessary
to address this apparent paradox between previous data and
the data by Okuno et al.11-12 Dietary vitamin A is presented
to the liver as retinyl esters within chylomicron remnants.
Parenchymal uptake and hydrolysis of retinyl esters yields
retinol, which combines with retinol-binding protein in the
endoplasmic reticulum and is secreted from the hepatocytes.
Most of the secreted RBP-bound retinol is then taken up by
the stellate cells and is re-esterified to retinyl ester.13 Intracellular binding proteins help maintain low concentrations of
free retinol because in excess this compound can partition
into membranes and disrupt normal structure and function.
Under normal circumstances, about 50% to 80% of vitamin
A is stored in the HSC (HSC) in mammals as retinyl esters.14
HSC are instumental in providing tissues and cells with optimal amounts of vitamin A despite huge fluctuations in daily
vitamin A intake.
Of the four cytoplasmic binding proteins that are specific
for retinol and retinoic acid, cellular retinol binding protein
type I (CRBP-I) is most prominent in HSC. Each stellate cell
in normal rats has been caulculated to contain Ç20 1 106
molecules of CRBP-I.14 CRBP-I directs retinol to the enzyme
lecithin:retinol acyltransferase (LRAT) for esterification. Retinol acyltransferase also is present in a relatively high concentration in HSC.15 CRBP-I seems to be rate limiting for retinol
uptake and storage by stellate cells:vitamin A-deficient rats
exhibit a marked reduction in stellate-cell CRBP-I and concomitantly reduced retinol uptake, esterification, and storage.11-12 CRBP-I may also facilitate the irreversible oxidation
of retinol to the more biologically active retinoic acid,12 but
the mechanism for oxidation of retinol has not been proven
for HSC. It is important to note that once retinoic acid is
formed, it cannot be reduced to retinol and stored as retinyl
esters.
The potent biological effects of vitamin A are, in most
cases, mediated by six different nuclear retinoic acid receptors,16 five of which have been identified in stellate cells.17
These receptors regulate gene expression by binding to short
DNA sequences (retinoic acid response elements) in the vicinity of target genes. The most important ligand for the
retinoid X receptor (RXR) subfamily (RXR-a, RXR-b, and
RXR-g) is the 9-cis isomer of retinoic acid, whereas both alltrans and 9-cis retinoic acids are the most important ligands
for the retinoic acid receptor subfamily (RAR-a, RAR-b, and
RAR-g).12,16 The receptors can also be activated by other
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1068 BLOMHOFF
HEPATOLOGY October 1997
natural and synthetic retinoids. An active research have elucidated a complex pattern of activation of retinoids, which
involve homodimerization of RXRs, heterodimerization between RARs and RXRs, as well as between RXRs and other
members of the nuclear receptor family, interaction of the
nuclear receptors with coactivators and cosupressors, activation of receptors by different retinol and retinoic acid metabolites, and activities of different types of responsive elements.12,16 This high degree of complexity is essential to
tightly regulate the activity of retinoids.
Although retinoids seem generally to inhibit liver fibrosis,
the data of Okuno et al.10 clearly suggest that there are conditions in which they stimulate fibrosis. It should be noted,
however, that the present study found no effect of retinoic
acids at concentrations below 100 nmol/L. Half of the maximal effect on the secretion of total TGF-b was seen at 400
nmol/L. The physiological concentration of retinoic acids in
most cells and body fluids may be as low as 10 nmol/L,
although the data on this are limited.11-12 Pharmacological
intake of concentrated forms of retinol or retinoic acid can
increase the retinoic acid concentration significantly.
The anti-proliferative effect of retinoic acid is more pronounced in HSC than in myofibroblasts.4-6 In addition, RARb and RXR-a messenger RNA and retinoic acid response
elements binding activity of nuclear extracts are reduced in
myofibroblasts compared with stellate cells,17 which is an
indication of impaired retinoic acid receptor signaling in activated myofibroblasts. Changes in nuclear retinoid receptors
could, therefore, play a key role in the observation by Okuno
et al.10; retinoids stimulate RARE-regulated genes such as
CRBP(I), RXR-a and RAR-b in stellate cells. The latter, complexed with retinoic acid, block the activity of AP-1 (the fos/
jun family), a transcription factor important for TGF-b. As
HSC lose retinoids, if their expression of RAR-b, RXR-a, or
CRBP changes, AP-1 activity may increase, leading to increased transcription of AP-1 responsive genes, such as TGFb and, thence, to fibrogenesis. As the stimulation of TGF-b
activation by retinoic acid occurs under circumstances in
which the tonic inhibitory effect of retinoids (which presumably is mediated via nuclear receptors) on fibrogenesis is
diminished, mechanisms apart from nuclear retinoic acid receptors, such as retinoylation18 or direct effects on membrane
proteins,19 might be involved.
At present, from the data available it may be postulated
that the retinyl ester storage is a key determinant in preserving the quiescent phenotype in stellate cells; however, this
remains to be proven. The newly discovered active retinolmetabolites (14-hydroxy-4, 14-retro-retinol, and 4-oxoretinol)20-21 are candidates for sensors of retinyl esters storage
that potentially could mediate such an effect. If this is the
case, retinoic acid, which cannot be converted to the active
retinol metabolites, does not have this possibility of protective action.
Although much remains to be learned, in linking retinoic
acid exposure to TGF-b activation, the study by Okuno et
al.10 adds a potentially important new dimension to the role
of vitamin A metabolites in liver injury.
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RUNE BLOMHOFF, Ph.D.
Institute for Nutrition Research
School of Medicine
University of Oslo
Oslo, Norway
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