Ciona Intestinalis Research Articles

Alternative respiratory chain enzymes: Therapeutic potential and possible pitfalls

The alternative respiratory chain (aRC), comprising the alternative NADH dehydrogenases (NDX) and quinone oxidases (AOX), is found in microbes, fungi and plants, where it buffers stresses arising from restrictions on electron flow in the oxidative phosphorylation system. The aRC enzymes are also found in species belonging to most metazoan phyla, including some chordates and arthropods species, although not in vertebrates or in Drosophila. We postulated that the aRC enzymes might be deployed to alleviate pathological stresses arising from mitochondrial dysfunction in a wide variety of disease states. However, before such therapies can be contemplated, it is essential to understand the effects of aRC enzymes on cell metabolism and organismal physiology. Here we report and discuss new findings that shed light on the functions of the aRC enzymes in animals, and the unexpected benefits and detriments that they confer on model organisms. In Ciona intestinalis, the aRC is induced by hypoxia and by sulfide, but is unresponsive to other environmental stressors. When expressed in Drosophila, AOX results in impaired survival under restricted nutrition, in addition to the previously reported male reproductive anomalies. In contrast, it confers cold resistance to developing and adult flies, and counteracts cell signaling defects that underlie developmental dysmorphologies. The aRC enzymes may also influence lifespan and stress resistance more generally, by eliciting or interfering with hormetic mechanisms. In sum, their judicious use may lead to major benefits in medicine, but this will require a thorough characterization of their properties and physiological effects.

Compared stress tolerance to short-term exposure in native and invasive tunicates from the NE Atlantic: when the invader performs better

The combined impact of invasive species and climate change threatens natural systems worldwide, often facilitating the expansion of harmful invasive species. It is imperative to understand the mechanisms behind why species become invasive and widespread. Traditionally, it is thought that invasive species have greater tolerances to a wider array of environmental conditions than natives. We, therefore, tested the hypothesis that invasive species are more tolerant to the effects of short-term exposure to temperature and salinity stress. Using unifactorial experiments, we compared the tolerances of two common fouling NE Atlantic ascidians, the native Ciona intestinalis and the invasive Styela clava, to increased temperature and decreased salinity. We measured lethal and behavioural responses affecting 50% of populations to give an indication of the tolerance limits for temperature (LT50) and salinity (EC50), and respiration rate to give an indication of the change in metabolic response. The invasive S. clava was more tolerant to increased stress (LT50 = 29.5 °C, EC50 = 19.5) compared with C. intestinalis (LT50 = 27.0 °C, EC50 = 22.7), whereas both species displayed similar metabolic responses observed through increased respiration rates. This study is among the first to experimentally determine limits for temperature and hyposalinity stress for either species and supports the hypothesis that the invader performs better under extreme conditions. Future environmental changes caused by events such as heat waves and climate change could push species to the edge of their physiological limits, potentially facilitating competitive shifts between native and invasive species.

Prevalence of Mutation-Prone Microhomology-Mediated End Joining in a Chordate Lacking the c-NHEJ DNA Repair Pathway

Classical non-homologous end joining (c-NHEJ), afundamental pathway that repairs double-strand breaks in DNA, is almost universal in eukaryotes and involves multiple proteins highly conserved from yeast to human [1]. The genes encoding these proteins were not detected in the genome of Oikopleura dioica, a new model system of tunicate larvaceans known for its very compact and highly rearranged genome [2-4]. After showing their absence in the genomes of six other larvacean species, the present study examined how O.dioica oocytes and embryos repair double-strand DNA breaks (DSBs), using two approaches: the injection of linearized plasmids, which resulted in their rapid end joining, and a newly established CRISPR Cas9 technique. In both cases, end joining merged short microhomologous sequences surrounding the break (mainly 4bplong), thus inducing deletions larger than for the tunicate ascidian Ciona intestinalis and human cells. A relatively high frequency of nucleotide insertions was also observed. Finally, a survey of genomic indels supports the involvement of microhomology-mediated repair in natural conditions. Overall, O.dioica repairs DSBs as other organisms do when their c-NHEJ pathway is experimentally rendered deficient, using another mode of end joining with the same effect as alternative NHEJ (a-NHEJ) or microhomology-mediated end joining (MMEJ) [5-7]. We discuss how the exceptional loss of c-NHEJ and its replacement by a more mutation-prone mechanism may have contributed to reshaping this genome and even been advantageous under pressure for genome compaction.

Regulatory cocktail for dopaminergic neurons in a protovertebrate identified by whole-embryo single-cell transcriptomics.

The CNS of the protovertebrate Ciona intestinalis contains a single cluster of dopaminergic (DA) neurons, the coronet cells, which have been likened to the hypothalamus of vertebrates. Whole-embryo single-cell RNA sequencing (RNA-seq) assays identified Ptf1a as the most strongly expressed cell-specific transcription factor (TF) in DA/coronet cells. Knockdown of Ptf1a activity results in their loss, while misexpression results in the appearance of supernumerary DA/coronet cells. Photoreceptor cells and ependymal cells are the most susceptible to transformation, and both cell types express high levels of Meis Coexpression of both Ptf1a and Meis caused the wholesale transformation of the entire CNS into DA/coronet cells. We therefore suggest that the reiterative use of functional manipulations and single-cell RNA-seq assays is an effective means for the identification of regulatory cocktails underlying the specification of specific cell identities.

Progenitor targeting by adult stem cells in Ciona homeostasis, injury, and regeneration

In the ascidian Ciona intestinalis, oral siphon amputation activates adult stem cell niches in the branchial sac to divide and dispatch migratory progenitor cells to a regeneration blastema at the site of injury. This study shows that progenitor cells derived from branchial sac stem cell niches have roles in homeostasis, wound repair, and regeneration of the siphons and neural complex (NC). During homeostasis, progenitor cells targeted the pharyngeal stigmata to replace ciliated cells involved in filter feeding. After individual or double siphon amputations, progenitor cells specifically targeted the oral or atrial siphons or both siphons, and were involved in the replacement of siphon circular muscle fibers. After oral siphon wounding, progenitor cells targeted the wound sites, and in some cases a supernumerary siphon was formed, although progenitor cell targeting did not predict the induction of supernumerary siphons. Following NC ablation, progenitor cells specifically targeted the regenerating NC, and supplied the precursors of new brain and neural gland cells. The tissues and organs targeted by branchial sac stem cells exhibited apoptosis during homeostasis and injury. It is concluded that branchial sac progenitor cells are multipotent and show targeting specificity that is correlated with apoptosis during homeostatic growth, tissue repair, and regeneration.

Bioenergetic consequences of xenotopic expression of Ciona intestinalis alternative oxidase (AOX) in the mouse

Bioenergetic consequences of xenotopic expression of Ciona intestinalis alternative oxidase (AOX) in the mouse

Comparative feeding rates of native and invasive ascidians

Ascidians have a recent history of species introductions globally, often with strong ecological impacts. Comparisons of per capita effects of invaders and comparable natives are useful to assess such impacts. Here, we explore ingestion rates (IR) and clearance rates (CR) of Ciona intestinalis and Ciona robusta, co-occurring native and non-native ascidians, respectively, from Brittany, France. IR was positively related to food concentration, with the invader responding more strongly to increasing food concentration. CR also differed by species, with the invader demonstrating higher values. C. robusta exhibited a higher functional response (Type I) than did C. intestinalis (Type II). Relative impact measured using seasonal abundance and IR revealed that C. robusta has a much greater impact than C. intestinalis at all food concentrations tested, though the former has a constrained distribution which limits its regional impact. Nevertheless, when abundant, we expect C. robusta to exert a greater impact on algal foods.

Shared evolutionary origin of vertebrate neural crest and cranial placodes.

Placodes and neural crests represent defining features of vertebrates, yet their relationship remains unclear despite extensive investigation1-3. Here we use a combination of lineage tracing, gene disruption and single-cell RNA-sequencing assays to explore the properties of the lateral plate ectoderm of the proto-vertebrate, Ciona intestinalis. There are notable parallels between the patterning of the lateral plate in Ciona and the compartmentalization of the neural plate ectoderm in vertebrates4. Both systems exhibit sequential patterns of Six1/2, Pax3/7 and Msxb expression that depend on a network of interlocking regulatory interactions4. In Ciona, this compartmentalization network produces distinct but related types of sensory cells that share similarities with derivatives of both cranial placodes and the neural crest in vertebrates. Simple genetic disruptions result in the conversion of one sensory cell type into another. We focused on bipolar tail neurons, because they arise from the tail regions of the lateral plate and possess properties of the dorsal root ganglia, a derivative of the neural crest in vertebrates5. Notably, bipolar tail neurons were readily transformed into palp sensory cells, a proto-placodal sensory cell type that arises from the anterior-most regions of the lateral plate in the Ciona tadpole6. Proof of transformation was confirmed by whole-embryo single-cell RNA-sequencing assays. These findings suggest that compartmentalization of the lateral plate ectoderm preceded the advent of vertebrates, and served as a common source for the evolution of both cranial placodes and neural crest3,4.

Feeding plasticity more than metabolic rate drives the productivity of economically important filter feeders in response to elevated CO2 and reduced salinity

AbstractClimate change driven alterations in salinity and carbonate chemistry are predicted to have significant implications particularly for northern costal organisms, including the economically important filter feeders Mytilus edulis and Ciona intestinalis. However, despite a growing number of studies investigating the biological effects of multiple environmental stressors, the combined effects of elevated pCO2 and reduced salinity remain comparatively understudied. Changes in metabolic costs associated with homeostasis and feeding/digestion in response to environmental stressors may reallocate energy from growth and reproduction, affecting performance. Although these energetic trade-offs in response to changes in routine metabolic rates have been well demonstrated fewer studies have investigated how these are affected by changes in feeding plasticity. Consequently, the present study investigated the combined effects of 26 days’ exposure to elevated pCO2 (500 µatm and 1000 µatm) and reduced salinity (30, 23, and 16) on the energy available for growth and performance (Scope for Growth) in M. edulis and C. intestinalis, and the role of metabolic rate (oxygen uptake) and feeding plasticity [clearance rate (CR) and absorption efficiency] in this process. In M. edulis exposure to elevated pCO2 resulted in a 50% reduction in Scope for Growth. However, elevated pCO2 had a much greater effect on C. intestinalis, with more than a 70% reduction in Scope for Growth. In M. edulis negative responses to elevated pCO2 are also unlikely be further affected by changes in salinity between 16 and 30. Whereas, under future predicted levels of pCO2C. intestinalis showed 100% mortality at a salinity of 16, and a >90% decrease in Scope for Growth with reduced biomass at a salinity of 23. Importantly, this work demonstrates energy available for production is more dependent on feeding plasticity, i.e. the ability to regulate CR and absorption efficiency, in response to multiple stressors than on more commonly studied changes in metabolic rates.

Developmental arrest in Drosophila melanogaster caused by mitochondrial DNA replication defects cannot be rescued by the alternative oxidase

The xenotopic expression of the alternative oxidase AOX from the tunicate Ciona intestinalis in diverse models of human disease partially alleviates the phenotypic effects of mitochondrial respiratory chain defects. AOX is a non-proton pumping, mitochondrial inner membrane-bound, single-subunit enzyme that can bypass electron transport through the cytochrome segment, providing an additional site for ubiquinone reoxidation and oxygen reduction upon respiratory chain overload. We set out to investigate whether AOX expression in Drosophila could counteract the effects of mitochondrial DNA (mtDNA) replication defects caused by disturbances in the mtDNA helicase or DNA polymerase γ. We observed that the developmental arrest imposed by either the expression of mutant forms of these enzymes or their knockdown was not rescued by AOX. Considering also the inability of AOX to ameliorate the phenotype of tko25t, a fly mutant with mitochondrial translation deficiency, we infer that this alternative enzyme may not be applicable to cases of mitochondrial gene expression defects. Finding the limitations of AOX applicability will help establish the parameters for the future putative use of this enzyme in gene therapies for human mitochondrial diseases.

Oceanographic barriers to gene flow promote genetic subdivision of the tunicate Ciona intestinalis in a North Sea archipelago

Pelagic larval development has the potential to connect populations over large geographic distances and prevent genetic structuring. The solitary tunicate Ciona intestinalis has pelagic eggs and a swimming larval stage lasting for maximum a few days, with the potential for a homogenizing gene flow over relatively large areas. In the eastern North Sea, it is found in a geomorphologically complex archipelago with a mix of fjords and open costal habitats. Here, the coastal waters are also stratified with a marked pycnocline driven by salinity and temperature differences between shallow and deep waters. We investigated the genetic structure of C. intestinalis in this area and compared it with oceanographic barriers to dispersal that would potentially reduce connectivity among local populations. Genetic data from 240 individuals, sampled in 2 shallow, and 4 deep-water sites, showed varying degrees of differentiation among samples (FST = 0.0–0.11). We found no evidence for genetic isolation by distance, but two distant deep-water sites from the open coast were genetically very similar indicating a potential for long-distance gene flow. However, samples from different depths from the same areas were clearly differentiated, and fjord samples were different from open-coast sites. A biophysical model estimating multi-generation, stepping-stone larval connectivity, and empirical data on fjord water mass retention time showed the presence of oceanographic barriers that explained the genetic structure observed. We conclude that the local pattern of oceanographic connectivity will impact on the genetic structure of C. intestinalis in this region.

Control of mitochondrial superoxide production by reverse electron transport at complex I

The generation of mitochondrial superoxide (O2˙̄) by reverse electron transport (RET) at complex I causes oxidative damage in pathologies such as ischemia reperfusion injury, but also provides the precursor to H2O2 production in physiological mitochondrial redox signaling. Here, we quantified the factors that determine mitochondrial O2˙̄ production by RET in isolated heart mitochondria. Measuring mitochondrial H2O2 production at a range of proton-motive force (Δp) values and for several coenzyme Q (CoQ) and NADH pool redox states obtained with the uncoupler p-trifluoromethoxyphenylhydrazone, we show that O2˙̄ production by RET responds to changes in O2 concentration, the magnitude of Δp, and the redox states of the CoQ and NADH pools. Moreover, we determined how expressing the alternative oxidase from the tunicate Ciona intestinalis to oxidize the CoQ pool affected RET-mediated O2˙̄ production at complex I, underscoring the importance of the CoQ pool for mitochondrial O2˙̄ production by RET. An analysis of O2˙̄ production at complex I as a function of the thermodynamic forces driving RET at complex I revealed that many molecules that affect mitochondrial reactive oxygen species production do so by altering the overall thermodynamic driving forces of RET, rather than by directly acting on complex I. These findings clarify the factors controlling RET-mediated mitochondrial O2˙̄ production in both pathological and physiological conditions. We conclude that O2˙̄ production by RET is highly responsive to small changes in Δp and the CoQ redox state, indicating that complex I RET represents a major mode of mitochondrial redox signaling.

Dimerization of the voltage-sensing phosphatase controls its voltage-sensing and catalytic activity.

Multimerization is a key characteristic of most voltage-sensing proteins. The main exception was thought to be the Ciona intestinalis voltage-sensing phosphatase (Ci-VSP). In this study, we show that multimerization is also critical for Ci-VSP function. Using coimmunoprecipitation and single-molecule pull-down, we find that Ci-VSP stoichiometry is flexible. It exists as both monomers and dimers, with dimers favored at higher concentrations. We show strong dimerization via the voltage-sensing domain (VSD) and weak dimerization via the phosphatase domain. Using voltage-clamp fluorometry, we also find that VSDs cooperate to lower the voltage dependence of activation, thus favoring the activation of Ci-VSP. Finally, using activity assays, we find that dimerization alters Ci-VSP substrate specificity such that only dimeric Ci-VSP is able to dephosphorylate the 3-phosphate from PI(3,4,5)P3 or PI(3,4)P2 Our results indicate that dimerization plays a significant role in Ci-VSP function.

Identification of CPE and GAIT elements in 3’UTR of macrophage migration inhibitory factor (MIF) involved in inflammatory response induced by LPS in Ciona robusta

Innate immune responses face infectious microorganisms by inducing inflammatory responses. Multiple genes within distinct functional categories are coordinately and temporally regulated by transcriptional ‘on’ and ‘off’ switches that account for the specificity of gene expression in response to external stimuli. Mechanisms that control transcriptional and post-transcriptional regulation are important in coordinating the initiation and resolution of inflammation. Macrophage migration inhibitory factor (MIF) is an important cytokine that, in Ciona robusta, is related to inflammatory response. It is well known that in C. robusta, formerly known as Ciona intestinalis, the pharynx is involved in the inflammatory reaction induced by lipopolysaccharide (LPS) injection in the body wall. Using this biological system, we describe the identification of two C. robusta MIFs (CrMIF1 and CrMIF2). The phylogenetic tree and modeling support a close relationship with vertebrate MIF family members. CrMIF1 and CrMIF2 possess two evolutionally conserved catalytic sites: a tautomerase and an oxidoreductase site with a conserved CXXC motif. Real-time PCR analysis shows a prompt expression induced by LPS inoculation in CrMIF1 and a late upregulation of CrMIF2 and in silico analyses of 3’UTR show a cis-acting GAIT element and a CPE element in 3’-UTR, which are not present in the 3’-UTR of CrMIF1, suggesting that different transcriptional and post-transcriptional control mechanisms are involved in the regulation of gene expression of MIF during inflammatory response in C. robusta.

PiRNA-like small RNAs are responsible for the maternal-specific knockdown in the ascidian Ciona intestinalis Type A

The mRNAs stored in eggs are crucial for embryogenesis. To address functions of maternal mRNAs, we recently reported the novel method MASK (maternal mRNA-specific knockdown), which we used to specifically knockdown maternal transcripts in the ascidian Ciona intestinalis Type A. In MASK, the cis element of a maternal gene is fused with eGFP or Kaede reporter gene, and the cassette is introduced into Ciona genome by transposon-mediated transgenesis. In eggs of the transgenic lines, the maternal expression of the gene whose cis element is used for driving the reporter gene is suppressed. The zygotic expression of the gene is not suppressed, suggesting that the MASK method can distinguish between maternal and zygotic functions of a gene. Here we investigated the cis and trans factors responsible for MASK results. In the ovaries in which knockdown of a maternal gene occurs, a number of antisense small RNAs are expressed that are complementary to the sequence of the knocked-down genes. We suspect that these antisense small RNAs are the factor responsible for MASK results. The antisense small RNAs have several features that are seen in PIWI-interacting RNAs (piRNAs), suggesting that MASK is likely to use a piRNA-mediated mechanism to knock down maternal mRNAs.

Depletion of Maternal Cyclin B3 Contributes to Zygotic Genome Activation in the Ciona Embryo

Most animal embryos display a delay in the activationof zygotic transcription during early embryogenesis [1]. This process is thought to help coordinate rapid increases in cell number during early development [2]. The timing of zygotic genome activation (ZGA) during the maternal-to-zygotic transition (MZT) remains uncertain despite extensive efforts. We explore ZGA in the simple protovertebrate, Ciona intestinalis. Single-cell RNA sequencing (RNA-seq) assays identified Cyclin B3 (Ccnb3) as a putative mediator of ZGA. Maternal Ccnb3 transcripts rapidly diminish in abundance during the onset of zygotic transcription at the 8-cell and 16-cell stages. Disruption of Ccnb3 activity results in precocious activation of zygotic transcription, while overexpression abolishes normal activation. These observations suggest that the depletion of maternal Cyclin B3 products is a critical component of the MZT and ZGA. We discuss evidence that this mechanism might play a conserved role in the MZT of other metazoans, including mice and humans.

Dynamics of two key maternal factors that initiate zygotic regulatory programs in ascidian embryos

In animal embryos, transcription is repressed for a definite period of time after fertilization. In the embryo of the ascidian, Ciona intestinalis (type A; or Ciona robusta), transcription of regulatory genes is repressed before the 8- or 16-cell stages. This initial transcriptional quiescence is important to enable the establishment of initial differential gene expression patterns along the animal–vegetal axis by maternal factors, because the third cell division separates the animal and vegetal hemispheres into distinct blastomeres. Indeed, maternal transcription factors directly activate zygotic gene expression by the 16-cell stage; Tcf7/β-catenin activates genes in the vegetal hemisphere, and Gata.a activates genes in the animal hemisphere. In the present study, we revealed the dynamics of Gata.a and β-catenin, and expression profiles of their target genes precisely. β-catenin began to translocate into the nuclei at the 16-cell stage, and thus expression of β-catenin targets began at the 16-cell stage. Although Gata.a is abundantly present before the 8-cell stage, transcription of Gata.a targets was repressed at and before the 4-cell stage, and their expression began at the 8-cell stage. Transcription of the β-catenin targets may be repressed by the same mechanism in early embryos, because β-catenin targets were not expressed in 4-cell embryos treated with a GSK inhibitor, in which β-catenin translocated to the nuclei. Thus, these two maternal factors have different dynamics, which establish the pre-pattern for zygotic genetic programs in 16-cell embryos.

New maresin conjugates in tissue regeneration pathway counters leukotriene D4-stimulated vascular responses.

Resolution of acute inflammation is governed, in part, by lipid mediator class switching from proinflammatory eicosanoids to specialized proresolving mediators, including a recently identified new pathway of mediators, termed maresin conjugates in tissue regeneration (MCTR), which includes MCTR1, MCTR2, and MCTR3. Here, we addressed whether each MCTR can impact the known vascular actions of cysteinyl leukotrienes. Leukotriene D4 (LTD4; 1.5 nmol/mouse) initiated vascular leakage in mouse cremaster vessels, which was reduced (>75%) by MCTR1 and MCTR2 (0.15 nmol each). With isolated Ciona intestinalis (sea squirt) primordial hearts, LTD4 (1-100 nM) induced negative inotropic action and lowered heartbeats 20-30%. Each MCTR (1-100 nM) prevented LTD4-reduced heart rates. With human cysteinyl leukotriene receptor-1 (CysLT1) expressed in CHO cells, each MCTR (10-100 nM) significantly reduced LTD4-initiated signaling. To assess the contribution of CysLT1 in the proresolving actions of MCTR, we carried out human macrophage (MΦ) phagocytosis. Each MCTR (0.1-10 nM) stimulated human MΦ phagocytosis of live Escherichia coli, whereas LTD4 did not stimulate phagocytosis. MCTR-activated phagocytosis was significantly blocked by a pharmacologic receptor antagonist (MK571). With both CHO-CysLT1 and human MΦs, each MCTR competed for specific [3H]-LTD4 binding with apparent lower affinity than LTD4. Thus, each MCTR functionally interacts with human CysLT1 to pharmacologically counter-regulate vascular responses and stimulate physiologic phagocytosis with MΦs.-Chiang, N., Riley, I. R., Dalli, J., Rodriguez, A. R., Spur, B. W., Serhan, C. N. New maresin conjugates in tissue regeneration pathway counters leukotriene D4-stimulated vascular responses.

Not-so-simple sieving by ascidians: re-examining particle capture at the mesh and organismal scales

The particle capture mechanisms of biological filters determine the particle spectrum that is ingested by filter-feeding animals. Although ascidian feeding has been extensively investigated, the organismal-scale fluid dynamics and mesh-scale particle-filter interactions are not fully characterized. Fluorescein dye visualization of flow through the branchial sac of the ascidian Ciona intestinalis showed organismal-scale flow was laminar and moved both parallel and perpendicular to the mucous mesh. Endoscopic investigations of Herdmania momus revealed the mesh-scale filtration process, including the pre-capture velocities, particle approach angles, and mesh behavior. The mesh speed was variable (range 0–0.4 mm s−1). To determine how particle shape affects hydrosol capture, Styela plicata was fed differently shaped polystyrene particles (ellipsoids and spheres); sampling the inhaled and exhaled water revealed that microellipsoids (0.3 × 0.7 µm) were captured at significantly lower efficiency (32%) than 1 µm microspheres (86%). The capture efficiency of microellipsoids resembled that of microspheres with a diameter similar to the microellipsoids’ minor axis (0.3 µm, 31%) suggesting that the minimum diameter of ellipsoidal particles determines the capture efficiency. Flow near the filter was parallel to the mesh even ~ 10 s of micrometers away, implicating a “crossflow” component to ascidian filtration, where the fluid being filtered is directed along the surface of the filter rather than exclusively perpendicular to it. Collectively, these results suggest that ascidian filtration acts as a hybrid-flow filtration system rather than a classical direct sieve.

Ciona intestinalis calcitonin-like peptide promotes osteoblast differentiation and mineralization through MAPK pathway in MC3T3-E1 cells

Abstract This study examined the effects of Ciona intestinalis calcitonin-like peptide (CCLP) on osteoblast differentiation and mineralization in the culture system of MC3T3-E1 cells. The primary structure of the CCLP containing Cys-Asp-Gly-Val-Ser-Thr-Cys-Trp-Leu-His-Glu-Leu-Gly-Asn-Ser-Val-His-Ala-Thr-Ala-Gly-Gly-Lys-Gln-Asn-Val-Gly-Phe-Gly-Pro-NH2 was synthesized automatically using the solid phase method with fluorenylmethoxycarbonyl (Fmoc) resin. Pre-osteoblast MC3T3-E1 cells were cultured with various concentrations of CCLP (7.5, 15, and 30 μM) during the osteoblast differentiation period. To examine osteoblast differentiation, alkaline phosphatase (ALP) activity was determined by reading the absorbance at 405 nm using a spectrophotometer, and mineralization was evaluated by staining with Alizalin red S. Moreover, the expression of differentiation markers such as ALP, osteocalcin, and osteopontin was measured using RT-PCR and Western blot analysis. The results showed that CCLP did not exhibit any cytotoxic effect on MC3T3-E1 cells even at the highest concentration (30 μM) at 2 and 5 days. CCLP also enhanced MC3T3-E1 cells proliferation, differentiation, and mineralization demonstrated by the increased expression of several osteoblast phenotype markers such as ALP, and Alizarin Red S staining. In addition, the CCLP induced mitogen-activated protein kinase (MAPK) pathway in MC3T3-E1 cells. These results suggest that CCLP exerts positive effects on osteoblast differentiation and may represent a potential target for pharmaceutical development.