Calponin in Non-Muscle Cells
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- Wu, K. & Jin, J. Cell Biochem Biophys (2008) 52: 139. doi:10.1007/s12013-008-9031-6
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Calponin is an actin filament-associated regulatory protein expressed in smooth muscle and non-muscle cells. Calponin is an inhibitor of the actin-activated myosin ATPase. Three isoforms of calponin have been found in the vertebrates. Whereas the role of calponin in regulating smooth muscle contractility has been extensively investigated, the function and regulation of calponin in non-muscle cells is much less understood. Based on recent progresses in the field, this review focuses on the studies of calponin in non-muscle cells, especially its regulation by cytoskeleton tension and function in cell motility. The ongoing research has demonstrated that calponin plays a regulatory role in non-muscle cell motility. Therefore, non-muscle calponin is an attractive target for the control of cell proliferation, migration and phagocytosis, and the treatment of cancer metastasis.
KeywordsCalponinCytoskeletonCell motilityMigrationProliferationPhagocytosisCancer metastasis
The function of calponin in smooth muscle contraction has been extensively investigated. Biochemical and contractility analyses demonstrated that calponin inhibits actin-activated myosin ATPase . Although the physiological function of calponin in smooth muscle in vivo is still inconclusive, the amino acid sequences of the three calponin isoforms are highly conserved in most parts of the polypeptide chain , suggesting that they may function by conserved mechanisms to regulate the actin filaments in smooth muscle and non-muscle cells. On the other hand, the three isoforms of calponin show significant structural differences in the C-terminal region and have distinct patterns of cell type-specific expression , indicating their functional diversities corresponding to the specific cellular environment and/or the activity of different cell types.
Extended from smooth muscle calponin studies, this review focuses on the role of calponin in non-muscle cell motility. Led by background information for the structure–function relationship of calponin, the regulation and function of h2-calponin in several non-muscle cell types will be discussed in detail in order to provide readers with an update for the new knowledge learned in the recent years.
Cell Type-Specific Expression of the Three Calponin Isoforms and Functional Implications
The three calponin isoforms with distinct charge properties show differentiated expression patterns in smooth muscle and in various types of non-muscle cells. The h1 (basic) isoform of calponin is specific to differentiated smooth muscle cells and up-regulated during post-natal development [9, 10], consistent with a role in contractile function. The expression of h2 (neutral) calponin shows a broader tissue distribution pattern, including developing and remodeling smooth muscles , epidermal keratinocytes, fibroblasts , lung alveolar cells , endothelial cells , and white blood cells of myeloid lineage . The h3 (acidic) calponin has been found in smooth muscle and non-muscle tissue  with a potential function in regulating actin filaments during neuronal remodeling . The function of h1-calponin in regulating smooth muscle contractility has been extensively investigated and the information established from h1-calponin studies laid a foundation for investigations on the function of h2 and h3 calponins in non-muscle cell motility.
Despite the in vitro experimental evidence that suggests a role of calponin in regulating the function of smooth muscle thin filaments [7, 15], the phenotypes of h1-calponin gene knockout mice indicated that the lack of h1-calponin does not abolish the contraction of smooth muscle  or the signal transduction of Ca2+ sensitization pathways in smooth muscle . This outcome supports earlier observations that vascular smooth muscle of Wistar rats is contractile, although it naturally lacks calponin [18, 19]. Whereas calponin is not an essential element for smooth muscle to contract, the contraction of rat aortic smooth muscle that lacks calponin showed decreased sensitivity to norepinephrine activation . Therefore, h1-calponin may play a modulator role in the function of smooth muscle actin filaments.
Consistent with its proposed role in fine-tuning of smooth muscle contractility, changes in h1-calponin expression have been found in physiological and pathological adaptations. For example, a high level of h1-calponin mRNA was detected in smooth muscle of gallbladder of pregnant guinea pigs in correlation to the dysfunction of extrahepatic biliary tract, bile stasis in gallbladder, and gallstone formation . The level of h1-calponin in gastrointestinal smooth muscle was decreased in mice fed with senna extracts to chronically increase gastrointestinal motility and elevated in a cirrhosis rat model with chronically depressed gastrointestinal motility .
The role of h2-calponin in smooth muscle remains to be investigated. Significant expression of h2-calponin is detected in the growing smooth muscle such as embryonic stomach and urinary bladder as well as uterus during early pregnancy . On the other hand, the expression of h2-calponin decreased to low levels in quiescent adult smooth muscle cells, indicating a function correlating to cell proliferation. Recent studies have found high levels of h2-calponin in non-muscle cells including epithelial cells [8, 11], endothelial cells , fibroblasts , and myeloid phagocytes . The diverse expression pattern of h2-calponin lead to a hypothesis that h2-calponin plays a function that is common in these non-muscle cell types, such as the regulation of the actin cytoskeleton.
Acidic (h3) calponin has been found in smooth muscle and neuronal tissues. The function of acidic calponin has not been extensively investigated. In vitro experiments showed that acidic calponin preferentially binds to F-actin than G-actin, and might be degraded by mu-calpain proteolysis . Although an early study did not detect any interaction between acidic calponin and Ca2+-calmodulin , a more recent study showed moderate (10−5 M range) binding affinity between acidic calponin and Ca2+-calmodulin . Together with the unique tissue distribution, the likely role of acidic calponin in the regulation of cytoskeleton function needs further investigation.
Structure–Function Relationships of Calponin
Protein binding studies have documented that calponin binds actin [1, 23, 24] and cross-links actin filaments . It also binds Ca2+-calmodulin [1, 2], tropomyosin , myosin [27, 28], the 20-kDa regulatory light chain of myosin , desmin [30, 31], tubulin , caldesmon , caltropin , gelsolin , and phospholipids .
Through high affinity binding to F-actin at two sites (Fig. 2b) , calponin inhibits (a) the actin-activated Mg-ATPase of smooth muscle myosin in vitro [38–40], (b) the movement of actin filaments over immobilized myosin in in vitro motility assays [41, 42], and (c) force development and shortening velocity in permeabilized smooth muscle strips with no effect on maintained rigor contraction [43, 44]. The association of calponin with actin filament and its inhibitory effects on actomyosin ATPase formed the basis of a hypothesis that calponin may represent a striated muscle troponin-like thin filament regulatory system in smooth muscle .
An increasing number of proteins have been found to contain structural similarities to a sequence motif of approximately 100 amino acids in calponin (e.g., residues 29–129 in chicken h1-calponin, Fig. 2b). The term “calponin homology (CH) domain” is used to describe this structure . CH domains have been found in a variety of proteins ranging from actin cross-linking to signaling and are proposed to function either as autonomous actin-binding motifs or to serve a regulatory function. Despite the overall sequence conservation of the CH domains, the individual modules display a great variability in function and proteins containing CH domain do not show apparent functional correlation to calponin or to each other. While the function of the CH domain remains to be further determined, h1-calponin has been found to bind to extracellular regulated kinase (ERK) 1 and 2 through the CH domain, suggesting a function in the ERK signaling of smooth muscle and non-muscle cells .
Three repeating sequence motifs are found in the C-terminal region of calponin. This repeating structure is conserved in all calponin isoforms across species. Illustrated in Fig. 2b, the first repeating motif overlaps with the second actin-binding site  and contains protein kinase C (PKC) phosphorylation residues Ser175 and Thr184 (see the following section). Similar sequences as well as potential phosphorylation sites are present in repeats 2 and 3 (Fig. 2b). It remains to be investigated whether these motifs have similar functions.
Post-translational Regulation of Calponin
Various actomyosin ATPase assay experiments have demonstrated that the biochemical function of calponin is regulated by phosphorylation . Calponin is co-immunoprecipitated with the mitogen-activated protein kinase (MAPK) and with PKC-ε from the extract of smooth muscle cells. Calponin phosphorylation may play a role in the PKC-ε induced, Ca2+-independent contraction of vascular smooth muscle . Calponin redistributes to the cell membrane during phenylephrine stimulation when MAPK and PKC-ε are targeted to the plasmalemma [49, 50].
In biochemical assays, the inhibitory effect of calponin on actomyosin ATPase can be alleviated by PKC-catalyzed phosphorylation  and restored following dephosphorylation by type 2A  and type 1  phosphatases. Calponin phosphorylation in smooth muscle increased rapidly in response to prostaglandin F2a concomitant with the increase in tension. PKC inhibitors inhibited the tension development as well as calponin phosphorylation . Rho-kinase was also found to phosphorylate calponin in vitro and inhibit the binding of calponin to F-actin .
Ser175 and Thr184 (Fig. 2b) have been identified to be the major phosphorylation sites in h1-calponin by PKC and calmodulin-dependent kinase II. Phosphorylation of Ser175 releases the inhibition of actomyosin ATPase by calponin . Consistently, phosphorylation or mutation of Ser175 alters calponin’s overall molecular conformation . This residue and flanking sequences are conserved in all three calponin isoforms. Therefore, Ser175 may also be the regulatory site in h2- and acidic calponins. These phosphorylation sites are in one of the segments mapped with actin-binding activity (Fig. 2b) , consistent with the observation that phosphorylation of Ser175 decrease the binding affinity of calponin to F-actin .
Ser175 and Thr184 are both located in the first repeating motif in the calponin polypeptide chain (Fig. 2b). Although the Ser175 position is conserved in repeats 2 and 3 and the Thr184 position is conserved in repeat 2, less phosphorylation of repeats 2 and 3 was found in comparison to that of repeat 1 . The functional significance of the three repeating motifs in the phosphoryaltion regulation of calponin needs to be further studied.
Despite the evidence for phosphorylation regulation of calponin function established by in vitro biochemical analyses, phosphorylated calponin is not readily detectable in vivo under physiological conditions . Therefore, the cellular mechanisms for phosphorylation to regulate the physiological function of calponin remain to be established.
Calponin and the Function of Actin Cytoskeleton
A growing body of evidence has indicated that calponin may play a role in the function of non-muscle cytoskeleton . A significant amount of calponin was found to co-localize with the cytoskeletal actin filaments . Platelet-derived growth factor (PDGF)-BB down-regulated h1-calponin to non-detectable levels in A7r5 smooth muscle cells and resulted in a 47% loss in alpha-actin stress fibers, affecting the phorbol-12,13-dibutyrate (PDBu)-induced podosome formation . A requirement of calponin in agonist-induced signal transduction in smooth muscle cells found in antisense RNA inhibition experiments suggests a contribution to the cytoskeleton-based cell signaling .
Calponin’s function in regulating actin–myosin interaction, myosin ATPase activity, and actin’s interaction with other structural and regulatory proteins suggests its role in the control of cell motility and cytokinesis. Consistently, forced over-expression of calponin in cultured smooth muscle cells and fibroblasts transfected with viral promoter-directed expression vectors inhibited the rate of cell proliferation and the completion of cell division [10, 62]. Supporting calponin’s function as a suppressor of cell proliferation, h1-calponin knockout mice displayed a premature onset of cartilage formation and ossification, increased post-natal bone formation, and accelerated healing of bone fractures .
Tropomyosin has been demonstrated to enhance actin polymerization  and the stability of actin filaments against cytochalasins . H2-calponin may regulate the function of actin filaments together with the function of tropomyosin as there was a positive correlation between the levels of tropomyosin and the transfective expression of h2-calponin . Consistently, a decreased level of tropomyosin was found in macrophages of h2-calponin knockout mice . In cells lacking endogenous calponin, transfective expression of h2-calponin resulted in increased tolerance of actin filaments to cytochalasin B, indicating decreased stability of the actin skeleton .
Regulation of h2-Calponin Expression by Mechanical Tension in the Cytoskeleton
The expression of h2-calponin is rapidly up-regulated during postnatal lung development coincident with the respiratory expansion of alveolar epithelial cells . Ex vivo experiments further demonstrated that the expression of h2-calponin in the cell is dependent on cell anchorage in adhesion culture and the stiffness of the substrate on which the cells were anchored. When epidermal keratinocytes or fibroblasts were prevented from attachment to plastic culture dish by continuous vibration, the cells had significantly decreased expression of h2-calponin . Cells cultured on low stiffness soft polyacrylamide gel substrates expressed significantly lower levels of h2-calponin in comparison to the high level h2-calponin in cells cultured on high stiffness hard gel substrate or on rigid plastic dish . The results demonstrate that mechanical tension in the cytoskeleton produced against the substrate stiffness  regulates h2-calponin gene expression.
Treatment of monolayer cultures of NIH 3T3 fibroblasts that normally express significant amounts of h2-calponin with blebbistatin, an inhibitor of non-muscle myosin II , to diminish the mechanical tension built in the actin cytoskeleton  significantly decreased the levels of h2-calponin protein  and mRNA  in comparison with the controls. These observations further support the hypothesis that the mechanical tension built in the actin filaments by myosin motors regulates h2-calponin gene expression.
Interestingly, chronic cyclic stretching of monolayer cells cultured on silicon rubber membrane did not increase but decreased the expression of h2-calponin . This observation suggests that after the cellular structure is remodeled during chronic cyclic stretching to fit the stretched dimension (to avoid damage of the cellular structure), the relaxation phase during the stretching cycles would periodically release cytoskeleton tension and lower the total amounts of mechanical tension sensed by the cell over time, which would result in the decreased level of h2-calponin expression .
Cytoskeleton Tension-Dependent Proteolytic Degradation of h2-Calponin
A rapid degradation of h2-calponin occurs in lung tissues after a period of deflation . However, h2-calponin was very effectively protected from the rapid degradation when post-mortal mouse and rat lungs were inflated with air to mimic their in situ expanded volume and incubated at 37°C for 6 h in comparison to that seen in the collapsed lung incubated side by side . No apparent degradation of other major proteins was detected in the collapsed lung by SDS-polyacrylamide gel electrophoresis, suggesting that the degradation of h2-calponin was a selective proteolysis as a cellular response to decreased cytoskeleton tension. This tension-regulated proteolysis in lung alveolar cells was reproducible in ex vivo tension-release experiments . Acute decrease of cytoskeleton tension in cultured alveolar cells by reducing the substrate dimension resulted in the degradation of h2-calponin . Blebbistatin treatment of cultured cells also produced acute h2-calponin degradation, indicating the critical role of myosin II motor function that builds tension in the actin cytoskeleton .
H2-Calponin and Mechanoregulation
All living cells respond to mechanical forces by gene regulation and protein modifications. Structural and functional remodeling of the actin cytoskeleton plays a major role in cellular responses to mechanical stimuli. The mechanical tension-regulated gene expression and protein degradation of h2-calponin may affect the function and dynamics of the actin cytoskeleton. Therefore, the regulation and function of h2-calponin may represent a mechanism for the actin cytoskeleton to respond to the mechanical environment of the cell.
Among the h2-calponin expressing cell and tissue types, the lung alveoli are experiencing repetitive tensile and compressive loadings during respiration, the vascular smooth muscle and endothelial cells are subjected to hemodynamic force of the blood flow, the epidermal keratinocytes are under various mechanical tension changes, tension induces adaptive changes in fibroblasts residing in the dermal connective tissue such as in fibrotic skin lesions , and substrate anchorage is required for macrophage differentiation . Therefore, the h2-calponin expressing tissue and cell types all appear being physiologically regulated by and responsive to mechanical stimuli, suggesting a function of calponin in regulating cellular interactions with the mechanical environment.
The conversion of chemical signals into mechanical responses in living cells has been clearly demonstrated as exampled by the process of activating myosin ATPase to generate contractile force in muscle. In contrast, the mechanisms for cells to sense and convert mechanical signals into biochemical regulations during cellular responses to mechanical stimuli are much less understood. The finding of mechanical tension regulation of h2-calponin gene expression and protein turnover provides an informative experimental system to address this fundamental question.
Physiological and Pathological Significance of Calponin in Non-Muscle Cells
Many regulatory and functional correlations to calponin have been observed in non-muscle cells with physiological and/or pathological significance. For example, the expression of h1-calponin gene was down-regulated during the differentiation of AR42 J-B13 pancreatic cells into insulin-producing cells induced by activin A, a member of the transforming growth factor β family, and hepatocyte growth factor whereas over-expression of h1-calponin suppressed the activin A-stimulated transcriptional activity . The expression of h1-calponin in renal mesangial cells (mesenchyme-derived multipotential cells that display a smooth-muscle-like phenotype during glomerular inflammation) was suppressed under conditions that cause glomerular nephritis. Treatment with anti-glomerular basement membrane antibody in h1-calponin knockout mice induced more severe nephritis than in wild type mice . H1-calponin-null mutant mice displayed increased bone formation associated with enhancement of bone morphogenetic protein responses , decreased number of neurons in the paraventricular nucleus of the hypothalamus correlating to changes in locomotor activity, and intake of food and water . H1-calponin expression induced by heparin contributed to the inhibited proliferation of myometrial and leiomyomal smooth muscle cells . TGF-beta1 induced the expression of h1-calponin in prostate fibroblasts and myofibroblasts during aging . 15-deoxy-delta(12,14)-prostaglandin J(2), a natural ligand for peroxisome proliferator-activated receptor, induced cell G1 arrest with increases in the expression of h1-calponin . Heterogeneous nuclear ribonucleoprotein complex K was found to interact with calponin and ERK1/2 during the cell cycle .
Significant levels of h2-calponin were found in developing vasculature of zebra fish embryos from 16 to 30 h post-fertilization. Diminished expression of h2-calponin tampered the proper migration of endothelial cells during the formation of intersegmental vessels, suggesting a function of h2-calponin in endothelial cell migration during vascular development . H2-calponin in epidermal keratinocytes may function in the organization of cytoskeleton at the cytoplasmic region of cell-to-cell junctions . The actin cytoskeleton plays a major role in cell motility that is essential for the function of phagocytes. High levels of h2-calponin are expressed in peripheral blood cells of myeloid lineage and regulated in coordination with the level of actin during monocyte–macrophage differentiation . To demonstrate the role of h2-calponin in regulating macrophage motility and phagocytosis, h2-calponin gene disrupted mice had reduced numbers of peripheral blood neutrophils and monocytes. H2-calponin-free macrophages had a higher rate of proliferation and faster migration than that of h2-calponin-positive cells, suggesting a faster extravasation of peripheral monocytes and neutrophils. H2-calponin-free macrophages had reduced spreading in adhesion culture together with decreased tropomyosin in the actin cytoskeleton. The lack of h2-calponin also resulted in significantly increased phagocytotic activity, suggesting a novel mechanism in the regulation of macrophage function .
Acidic (h3) calponin is found in the brain, mainly expressed by neurons , astrocytes , and glial cells  in the actin cytoskeleton with a proposed role in the plasticity of neural tissues [82, 83]. Acidic calponin was also found in the post-synaptic side of symmetric synapses and accumulated in the post-synaptic densities of asymmetric synapses . The post-synaptic localization of acidic calponin was associated with actin filaments to increase their stability and correlated to enhanced frequency of miniature excitatory post-synaptic currents . These observations suggest that acidic calponin may play a role in regulating the morphology and differentiation of cells in the nervous system, presumably via the regulation of reorganization and dynamics of the actin cytoskeleton. In addition, the expression of acidic calponin, as well as h1-calponin, was down-regulated after PGF2α treatment of rat uterine decidual cells, suggesting their roles in the maintenance of normal pregnancy .
Calponin and Cancer
Consistent with calponin’s functions in regulating cell proliferation, migration, and vascular construction, a number of reports have suggested its role in the growth and prognosis of cancer. The expression of h1-calponin in the vasculature of tumor tissues was found to correlate with multiple pathological factors such as tumor type, size, grade, stage, infiltration, and proliferating indexes in renal cell carcinoma , renal angiomyolipoma , and colon cancer . Reduced expression of h1-calponin was found in the vascular smooth muscle inside melanoma lesions . On the other hand, the loss of h1-calpnonin in host mural cells prevented maturation of tumor vasculature in h1-calponin knockout mice. In vitro studies showed that the platelet-derived growth factor B-induced vascular smooth muscle migration was down-regulated by h1-calponin-deficiency, and forced expression of h1-calponin restored migration . Mice deficient in h1-calponin also exhibited fragility in blood vessels, peritoneal membranes and tissues when receiving i.v. inoculations of B16 melanoma cells .
H1-calponin was found in human osteosarcoma cells with the level positively correlated with the prognosis, in which the survival rate of patients whose tumors expressed h1-calponin was significantly higher . Reduced levels of h1-calponin have been found in leiomyosarcoma of the uterus , hepatocellular carcinoma , basal-like breast carcinoma , metastatic basal cell carcinomas , and prostate cancer [96, 97]. High throughput technology to detect the expression pattern of specific genes in patient samples allows a subclassifcation of tumors as calponin-positive and calponin-negative and helps to evaluate treatment responsiveness and predict prognosis .
DNA hypermethylation of a 5′ CpG site in exon 1 of h1-calponin gene accompanied the decreased expression of h1-calponin in human sarcoma tissues and cell lines . Transfective expression of h1-calponin significantly inhibited anchorage-independent tumor cell growth and in vivo tumorigenicity , possibly through regulating cytoskeletal activities  and inhibiting VEGF expression and angiogenesis in vivo by reducing cell proliferation and motility . Zinc deficiency was found to correlate with h1-calponin degradation in precancerous esophageal tissues . Adenovirus-mediated h1-calponin gene therapy against peritoneal dissemination of ovarian cancer showed bifunctional therapeutic effects on stabilizing peritoneal cytoskeleton to resist cancer cell invasion and on inhibiting tumor cell invasion and growth .
Despite the fact that h2 and h3 (acidic) calponins are found in multiple non-muscle cell types, their involvement in cancer cell biology has not been extensively investigated. It was found that TGF-beta up-regulates the expression of actin-binding proteins including h2-calponin, high-molecular-weight tropomyosins, and alpha-actinin . H2-calponin also plays a role in inhibiting the proliferation and migration of prostate cancer cells and its down-regulation in cancer cells correlates with the potency of metastasis . Similar to the cell anchorage-dependent expression of h2-calponin in fibroblasts and epidermal keratinocytes , proteomic analysis detected decreased expression of acidic calponin in spheroids versus monolayer cultures of COGA-5 colon cancer cell line . More cancer cell types need to be examined for changes in h2 and acidic calponins and the pathological significance. To understand the regulation and function of calponin in cancer cells will also help to understand the effects of tissue mechanical environment on tumorogenesis and metastasis.
In conclusion, three isoforms of calponin are present in smooth muscle and non-muscle cells to function as regulators of the actin cytoskeleton. Based on biochemical and physiological studies of smooth muscle calponin, recent experimental data demonstrated cytoskeleton tension-regulated gene expression and turnover of calponin with functional effects on the stability of the actin filaments. Further investigations into the regulation and function of calponin in non-muscle cell proliferation and migration under physiological and pathological conditions will not only provide fundamental knowledge for the regulation of actin-based cell motility, but also help to understand the development and treatment of diseases, such as wound healing, immune responses, and cancer metastasis.
Our research that contributed to the data discussed in this review article was supported by grants from the Medical Research Council of Canada, the Alberta Cancer Board, the American Heart Association, the March of Dime Birth Defects Foundation, and the National Institutes of Health (AR039750-130037 and HL086720) to J.-P. Jin.