Download Cell Specific Signal Transduction Pathways Regulating Na,K

Articles in PresS. Am J Physiol Cell Physiol (November 19, 2008). doi:10.1152/ajpcell.00553.2008
Cell Specific Signal Transduction Pathways Regulating Na,K-ATPase. Focus on “Shortterm effects of thyroid hormones on the Na+-K+-ATPase activity of chick embryo
hepatocytes during develop: focus on signal transduction.”
Jianxun Lei, Maneesh Bhargava, and David H. Ingbar
Departments of Medicine, Pediatrics, and Cellular & Integrative Physiology
University of Minnesota School of Medicine, Twin Cities
Minneapolis, MN
Address for Correspondence:
David H. Ingbar
University of Minnesota Pulmonary
MMC 276
420 Delaware St SE
Minneapolis, MN 55455
Email: [email protected]
Phone: 612-624-0999; Fax 612-625-2174
Copyright © 2008 by the American Physiological Society.
The Na,K-ATPase is a complex integral membrane protein that carries out active transport of
sodium and potassium across the cell plasma membrane, maintaining the ionic gradients.
Each subunit has multiple isozymes that are expressed in tissue and developmental specific
fashion. In addition to preservation of electrolyte and fluid balance in cells, organs and the
whole body, Na,K-ATPase also is a signal transducer and modulator of growth, apoptosis,
cell adhesion and motility [1]. Na,K-ATPase is involved in a broad range of important
physiological and pathological conditions, such as alveolar fluid clearance, renal sodium
homeostasis, cardiac function, embryonic heart development. As much as 25-30% of
cellular ATP can be utilized by Na,K-ATPase in maintenance of ion transport in transporting
epithelia. Given these key roles, it is not surprising that Na,K-ATPase is regulated at many
biochemical levels (transcription, translation, post-translational modification of protein,
protein distribution, kinetic activation and recycling) and by a wide variety of agents such as
hormones, growth factors and other molecules. One classic regulator in many tissues is
thyroid hormone, which was shown to stimulate Na,K-ATPase transcription in selected
tissues many years ago [11].
Thyroid hormones play fundamental roles in regulation of normal cell functions though two
distinct mechanisms: genomic and nongenomic actions. In the genomic action, thyroid
hormone interacts with its nuclear receptors (TRα and TRβ) and coregulatory factors
(coactivators and corepressors) and thus regulates the expression of target genes. Until
recently, many investigators argued that virtually all of thyroid hormone action occurred
through transcriptional effects, but over the past decade there has been growing recognition
that this is not the case. Thyroid hormones can generate rapid nongenomic biological
responses independent of gene transcription [4, 6]. Most of these effects have been described
for 3,5,3’ triiodo-L-thyronine (T3) [2], but they also can occur with T4 or other related
byproducts, including 3,5-diiodo-L-thyronine (3,5-T2) [12] and 3-iodo-L-thyronamine [17].
T3 can regulate Na,K-ATPase through either a genomic and/or nongenomic manner. T3
responsive elements exist in the 5’-flanking region of Na,K-ATPase α and β subunits [8].
Thyroid hormones regulate gene expression of Na.K-ATPase developmentally in many
systems. The postnatal increases in Na,K-ATPase expression in kidney, brain, and lung
depend on normal thyroid hormone status. The expression of Na,K-ATPase α3 isoform in
ferret myocardium is responsive to T3 in newborns younger than 3 wk of age, but not in
mature ferrets older than 6 wk of age or in the adult ferret heart [5] . In the developing rat
brain, thyroid hormone increases Na,K-ATPase activity and α-subunit protein in newborn,
but not adult, rats [18]. Brain Na,K-ATPase activity and α-isoform proteins are sensitive to
T3 as late as postnatal day 15, but after postnatal day 22, they no longer respond to T3. In
contrast, in the lung epithelium, Na,K-ATPase responsiveness to T3 is absent in midgestation and is acquired in the late gestational period [16]. Adult and late fetal lung
epithelial cells respond to T3 with a rapid, nongenomic increase in Na,K-ATPase activity and
in the quantity of Na,K-ATPase protein in the plasma membrane, but this response is absent
in fetal distal lung epithelial cells earlier than 19 days of gestation [11].
Incerpi and colleagues have characterized the effect of thyroid hormones on Na,K-ATPase
activity on chick embryo hepatocytes during development. This response is believed to be
linked to T3 effects on hepatocyte proliferation and differentiation [12]. The chick embryo
hepatocytes respond to T3 with a very rapid, but transient, decrease in Na,K-ATPase activity
followed by an increase in Na/H exchanger activity. In the present report, Scapin et al
showed that T3 transiently inhibited Na,K-ATPase activity in chick embryo hepatocytes at 14
and 19 days, with more potent, but shorter-lived, inhibition in the embryonic day 19 cells.
The day 19 hepatocytes had almost two-fold more basal Na,K-ATPase activity (without T3)
than the day 14 cells. Scapin et al found that T3 conjugated to agarose beads - making the
T3 unable to enter the cells - mimicked the inhibition of the Na,K-ATPase activity by free
T3, demonstrating that T3 initiates this response at the plasma membrane. 3,5-Diiodo-Lthyronine (3,5-T2), but not L-thyroxine (T4), also inhibited Na,K-ATPase activity. The
authors then sought to characterize some of the signal transduction pathways required for this
rapid, nongenomic response to T3. In embryonic day 14 hepatocytes, the T3 effect on Na,KATPase required protein kinase A (PKA), but not phosphatidylinositol 3-kinase (PI3K) or
protein kinase C (PKC). In contrast, for embryonic day 19 hepatocytes, the T3 effect
required activity of PKA, PI3K and PKC. Another age-dependent difference was that the
PI3K and PKC inhibitors reduced basal Na,K-ATPase activity (in the absence of T3) in the
day 14, but not the day 19, hepatocytes. This suggests that at day 14, but not day 19, PI3K
and PKC both maintain basal Na,K-ATPase activity, but at day 14 their activity is not needed
for the inhibition of sodium pump by T3. Taken together, these data suggest that the signal
transduction mechanisms involved in the T3 response change during embryonic maturation
of the hepatocytes. However, the relationships between the required activities of PKA, PKC
and PI3K in the day 19 response are not defined in this system. Further studies with other
methods of activating or inactivating the kinases and measuring their activities more directly
may elucidate their roles and interdependence.
It is interesting to contrast the Na,K-ATPase response and signal transduction pathways of
the embryonic hepatocytes with that of adult and fetal lung epithelial cells. First, in the adult
alveolar epithelial cells, T3 stimulated Na,K-ATPase within 30 minutes and with a maximum
effect after 6 hours – even without any transcriptional effect (due to the presence of
actinomycin D) [15]. Thus the non-genomic effect in the lung cells is much more sustained
compared with the very transient effect in the hepatocytes. Second, the signal transduction
pathways by which T3 alters Na,K-ATPase differ somewhat in the lung epithelium and
embryonic hepatocytes. T3 augments Na,K-ATPase activity in the lung cells by increasing
the amount of Na,K-ATPase protein in the plasma membrane. Inhibitors of Src kinase, PI3K
or the mitogen activated protein kinase (MAPK) ERK1/2 blocked the T3-stimulated effects
on Na,K-ATPase [13, 14]. In the absence of T3, overexpression of constitutively active
mutants of either PI3K or Src kinase increased Na,K-ATPase activity and its cell surface
expression [14]. Thus the T3 effect in the lung requires activation of two pathways: the
ERK1/2 pathway and the Src Kinase - PI3K pathway. Another difference in the involved
pathways is that in the adult lung epithelium inhibitors of PKA (H-8) and PKC
(bisindolymaleimide) do not block the T3-induced increase in Na,K-ATPase activity [14].
During alveolar epithelial development the signal transduction pathways of the fetal day 19rat cells are the same as those in the adult cells. Fetal day 18 cells – which do not respond to
T3 with an increase in Na,K-ATPase activity – do have increased Na,K-ATPase activity
when PI3K is constuitively over expressed [16]. These data suggest that acquisition of T3sensitive Na,K-ATPase stimulation by rat alveolar epithelial cells at fetal day 19 is associated
with maturation of the Src Kinase - PI3K – Akt pathway at the PI3K or a more proximal step
in the pathway.
The site of most nongenomic actions of T3 has been localized to receptors at the plasma
membrane receptor or in cytoplasm [6]. These receptors may be distinct molecules or may be
the classical nuclear thyroid hormone receptors TRα and β acting in a different location. In
HeLa and CV-1 cells thyroid hormone (T4 or T3) activates the mitogen-activated protein
kinases by binding to the integrin αVβ3 located in the plasma membrane [3]. MAPK/ERK
transduces the hormone signal into complex cellular/nuclear events, including angiogenesis
and tumor cell proliferation [6]. Alternatively, the conventional TR also have been reported
to bind the p85 α-subunit of PI3K and thus account for activation of PI3K kinase by T3 [19].
In a rat pituitary cell line (GH4C1), T3 simulates activity of KCNH2 channels already at the
plasma membrane through PI3K by increasing the binding of TR β located in the plasma
membrane to p85 α-subunit, the TR α does not appears to be involved in this process [19].
In addition, a mutant TR β activates PI3K signaling via protein-protein interaction in both the
cytoplasm and the nucleus in mouse thyroid [9]. In pancreatic β cells, T3 also activates Akt
via action of the TRβ1 receptor [20]. In contrast, TR α1 is the predominant TR isoform in
vascular endothelial cells and treatment of endothelial cells with T3 increased the association
TRα1 with the p85 α subunit of PI3K, leading to the phosphorylation and activation of Akt
and endothelial nitric oxide synthase (eNOS). TR α1, but not TR β1, interacts with PI3K in a
ligand-dependent manner in vascular endothelial cells [10]. More detailed experiments are
needed to identify whether T3 is interacting with the integrin αVβ3, TR α or β, or other
receptors in the embryonic hepatocyte model. We are early in our understanding of the
upstream molecular events involved in the rapid non-genomic effects of thyroid hormones.
The difference in T3’s effect on Na,K-ATPase activity (stimulation in lung epithelium,
inhibition in embryonic hepatocytes) and the different signal transduction pathways involved
emphasizes once again the complexity and highly cell specific regulation of Na,K-ATPase.
The cell specific impact of Na,K-ATPase phosphorylation by PKA and PKC has been widely
recognized [7]. It seems that the cell specificity extends to other components of the signal
transduction pathways regulating the sodium pump also.
The studies of Scapin and colleagues add to the growing literature demonstrating the
importance of non-genomic effects of thyroid hormones, also raising interesting questions
about the signaling pathways involved and their maturation with development. These studies
also emphasize the high degree of cell and developmental specificity of the signal
transduction pathways that influence Na,K-ATPase activity. This specificity makes it
challenging to understand the regulation and to develop generic ways to alter Na,K-ATPase
activity, but also provides an opportunity to target responses in specific cell types to up- or
down-regulate activity of the sodium pump.
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FIGURE LEGEND
Figure 1: Schematic of Signal Transduction Pathways by which T3 may alter Na,K-ATPase
Activity. The pathway for T3 alteration of Na,K-ATPase can be divided into 3
components: initiation; signal transduction and sodium pump activation. Data
suggest that T3 can act at the plasma membrane (without necessarily entering the cell)
to trigger non-genomic signal transduction pathways. T3 may bind to integrin
subunits, directly to thyroid hormone receptors (TR) or to other, as yet unidentified,
molecules in the plasma membrane. T3 also may enter the cell through thyroid
hormone transporters or diffusion and act in the cytoplasm to trigger these pathways
(not shown). In the second component, the activation of cytoplasmic signal
transduction pathways is highly cell specific (indicated by dashed lines, since the
precise steps are not defined). In different systems this involves the Src Kinase –
PI3K – Akt pathway; PKA; PKC; and/or MAP kinases, such as ERK1/2. The kinases
may act to phosphorylate Na,K-ATPase directly and alter either its activity or
translocation, or their effects may be indirect through additional steps. Recent data
suggests that there are interactions between the kinase pathways as well. The detailed
steps involved in these complex pathways are just beginning to be elucidated. In
addition, the directionality of the effect of T3 and kinase action on Na,K-ATPase
(stimulation or inhibition) varies in different cell types. The third component is the
change in activity of Na,K-ATPase. In some cells, such as alveolar epithelium,
stimulation occurs at least partially through increases in the quantity of plasma
membrane Na,K-ATPase protein, presumably due to increased translocation from
existing intracellular pools. In other cells, such as the embryonic hepatocytes studied
by Scapin et al, the mechanism of inhibition is not defined. In the schematic, physical
translocation of thyroid hormone receptors between nucleus, membrane and
cytoplasm or Na,K-ATPase between plasma membrane and cytoplasmic endosomal
pools is indicated by heavy solid lines. The bracketing of PKA, PKC and ERK1/2
does not indicate that these 3 enzymes are regulated in parallel, rather that they each
are potentially involved in the T3 signal transduction pathway in some cell types.
thyroid hormone receptors
TRD
TRE
integrin
E3
Dv
Src
PI3K
Akt
ERK1/2
PKC
PKA
Na,K-ATPase
TRE
TRD